Categories
Free Essays

Free Detection and Identification of Bacteria in Food

Rapid detection and identification of bacteria in food and clinical laboratories

Abstract

Modern technological progress has affected how microbiology is practiced. There is emphasis on the minimalisation of laboratory costs, cost-efficiency and reliability of tests for efficient bacterial identification from food cultures. Before using any technology, it is recommended that the products’ performance characteristics be first tested, particularly as theses characteristics, are often not determined by the manufacturers. Consequently, the sensitivity and specificity, amongst other factors, associated with the use of these tests will also not have been determined. Additional factors would benefit from the use of controls, such as in the form of large scale and controlled clinical trials, in order to study the products’ performance. It is to be borne in mind that the involvement of ‘rapid’ tests, including an enzyme-linked immunosorbent assay, in bacterial detection may serve bests as methods for expeditious detection and screening than for the purposes of confirmation.

1. Introduction

In order to help diagnose infectious diseases, such as the bacteria Salmonella, a leading cause of food poisoning, the need for specialised microbial tests has arisen. Testing food products using rapid methods is a complicated process requiring the balance of sensitivity and specificity for the achievement of a reliable result. The following sections will discuss the use of five different detection methods, flow cytometry, the enterotube II system, chromogenic media, the Enzyme linked immunoassay and polymerase chain reaction and the necessity to balance the specificity and sensitivity of each technique, for the most accurate means of bacterial detection.

2.0 Flow Cytometry

Flow cytometry (FCM) is based on the principles of excitation of light, light scattering and fluorochrome molecular emission for the purposes of generating data covering a number of different parametric readings. FCM focuses on cells that measure 0.5um to 40 ?m in diameter. The technique of FCM relies on the provision of a light source, which, are usually lasers, and the cells must first be covered in a layer of phosphate buffered saline before being able to intercept the focused source of light. In this technique, a sample, containing the cells being tested, are injected into the centre of a sheath flow. Flow cytometry provides an analysis of cellular interactions at the macromolecular level. FCM is a technique that is considered to be a critical component of research in the biomedical field (Nolan & Sklar,1998).

2.1 Milk testing

FCM is one technique which may be useful when testing the safety and quality of milk. Testing milk requires analysis of somatic cell count and microbial analysis. Tests have shown (Gunasekera, et al., 2003) that the analysis of milk, where a known number of cells have been inoculated, upon clearing can be performed by FCM. FCM is able to give a good indication of the somatic cell count in raw milk and when coupled with other methods such as techniques involving fluorescence staining, can be used in testing biological milk quality. This therefore has an important application in the dairy industry, particularly in quality testing.

2.2 Analysis of Water Quality

The use of flow cytometry has to date also occurred in tandem with heterotrophic plate count (HPC) for the rapid detection of the bacterial count of potable as well as raw water (Hoefel, et al., 2005). The results showed that FCM was much quicker than HCP, in detecting viable bacteria in samples that were classed as viable but not amenable to culture. The FCM method detected bacteria within an hour as opposed to several days, for the HCP technique.

Studies have tested the sensitivity of FC-based assays in comparison to the plaque assay method, to measure levels of an infection virus in a sample (Cantera, et al., 2010). Poliovirus infection (PV1) was tested and the FCM method applied to a water sample infected with PV1-infected cells. The study revealed that a combination of flow cytometry, used with fluorescence resonance energy transfer technology, is able to sensitively and quickly detect the presence of infectious virus in a sample of environmental water.

2.3 Specificity of FCM

FCM has also been used to investigate whether T4 phage infected cells with E. coli ATCC 111303 can be differentiated from uninfected cells, based on phage DNA fluorescent detection. The technique, involving the lysis of bacterial cells by phage, allowed for the detection for infected cells 35 minutes post infection. Thus, FCM is able to be specific, when used combined with phages of predetermined host specificity. Overall, FCM is able to quantitatively measure and sensitively detect molecular level interactions and as such it may be considered to be a robust and adaptable technology (Nolan & Sklar, 1998).

3.0 The enterotube ll system

The Enterotube II was described for the first time in 1969 (Painter & Isenberg, 1973). This technology is an example of a rapid system of multi-test nature, functioning as a biochemical and enzymatic test method. The test system, functions by identifying unclassified gram-negative, rod shaped and oxidase-negative bacteria, belonging to the family Enterobacteriaceae. The test is often conducted within clinical laboratories. The machine comprises a flat-sided tube within which are 12 compartments, developed to allow different biochemical tests to be conducted. The system does consistently produce accurate results, and hence is liable to produce occasional false results.

3.1 Sensitivity and specificity

Reports such as the one by Dalton et al., (1993) in the detection of bacteriuria, have found that upon screening, only 55% specificity and 93% sensitivity have been obtained. O’Hara (2005) reports that it may be valuable for the diagnostic laboratory running tests, using equipment such as the Enterotube II system, to first stipulate what levels of ‘accuracy’ and ‘discrimination’ they consider are acceptable from their systems of identification. Accuracy of identification may be maximised by using the skills of a qualified microbiologist to confirm the bacterial classification (O’Hara, 2005). An additional way to potentially maximise sensitivity and specificity is to send an isolate to a reference laboratory in order to confirm identity. Use of enterotube II system will be for the testing of oxidase-negative bacteria and hence it should first be established that the oxidase test is not positive. To achieve this, and improve the specificity, an oxidase test may be performed on the relevant cultures.

In order to improve interpretation of results from use of the Enterotube II system, a suitable incubation time should be used, such as 16 hours (in the analysis of carbohydrate reactions (Woolfrey, et al., 1981). Furthermore, tests resulting in ambiguous classifications should be reevaluated (Woolfrey, et al., 1981) in order to improve specificity, without hampering the tests’ sensitivity.

4.0 Chromogenic Media

Chromogenic media (or fluorogenic media) are a microbial growth media of microbial nature. The media contains enzymes that are linked to either fluorogen (involved in light reaction) or chromogen (involved in colour reaction) or a combination of both. The method works by detecting activities that are enzymatic in nature, that are produced by the target microorganisms. Enzymatic activities are detected by the use of either organic compounds or dyes, as microorganisms, which grow in the proximity of these compounds are liable to make a distinctive pattern of colouring or alternatively fluoresce, which can be detected under UV light. Chromogenic media were first designed for application in clinical settings, but have proven to be useful in food testing.

4.1 Sensitivity and specificity of chromogenic media

Chromogenic media are considered to be a sensitive method of media analysis, when compared to more conventional types of media analysis (Downes, 2001). This is because the chromogenic media method allows for a faster analysis, with a turnover time of 24 hours, and it is also considered to have a higher sensitivity. In the identification of E.coli or Listeria monocytogenes, for example, specially designed chromogenic media are available for the purposes of improving test sensitivity.

When considering Salmonella detection, a number of specialised chromogenic media that are able to improve the specificity of detection are available. A study by Perez et al., (2003) showed that both broth enrichment and increasing the incubation time by a factor of two (from 24 hours to 48 hours) effectively increases the sensitivity of all of the media being used. Furthermore, due to the specificity of the chromogenic media, (determined to be greater than 84% following a two-day incubation period), a reduction in the need to undergo confirmatory tests improved the overall sensitivity of the specialized chromogenic media. A second study by Monneri et al., (1994), for the comparison of two new types of agar, media of chromogenic nature, Salmonella Detection and Identification Medium (SMID) and Rambach agar, against two conventional types of media for the detection of Salmonella. The results revealed that the newer chromogenic agar media were notably more specific than the more conventional media. Rambach agar was furthermore slightly more specific than SMID, being able to detect all Salmonella serotypes following a complementary C8 esterase test. Hence, sensitivity and specificity can be maximised by increasing culture time to 2 days fully, and using Rambach agar where appropriate, such as in the detection of Salmonella serotypes.

5.0 Enzyme Linked Immunoassay

The Enzyme linked immunoassay (ELISA) is a common antibody based technique designed for microorganism, or pathogenic, detection. The method is noted to have a high standard of specificity and sensitivity (Evans et al., 1989). A quantitative, or qualitative method may be used for the purposes of interpreting the results, which are, respectively, via the use of an instrumental read-out or through visual means. Specialised test kits to aid in the detection of Listeria, Salmonella and other microorganisms are commercially available.

5.1 Sensitivity and specificity

A study by Evans et al., (1989) utilised ELISA in the detection of Campylobacter pylori. The specificity and sensitivity of the test allowed for the detection of serum immunoglobulin G (IgG) antibodies targeted against the cell-associated proteins of C. pylori. Values of specificity and positive predictive value were revealed to be 100% for the high molecular weight cell-associated proteins. Furthermore, the assay sensitivity was measured at 98.7%, with the negative predictive value recorded as 98.6%. This indicates that specialised ELISA tests are likely to be valuable in such instances as in the detection of H. pylori. Furthermore, the costs of using the ELISA, as noted by Evans et al., (1989) are that it is cost effective and readily usable, with a lower likelihood of obtaining false negatives than with other tests, such as the use of a ‘urea breath test’ which is also amenable to be useful for the same purpose.

Svennerholm & Holmgren, (1978) report that E. Coli can be sensitively detected using a ganglioside ELISA. The method was deemed to be reliable and allow a high level of reproducibility. In general, it has been reported that the specificity and, or, sensitivity of assays that are commercially available, such as the ELISA may be maximised by having set cut-off values decreed by the manufacturers, according to the target disease (Cuzzubbo, et al., 1999). Furthermore, the IgG test, due to having 100% specificity, is highly likely to be reliable, as a method for bacterial testing.

6.0 Polymerase Chain Reaction

Similar to the ELISA test, the ‘PCR’ or the polymerase chain reaction (PCR) is one of the most readily recognised and used diagnostic tool currently in use. PCR works by identifying a highly specific sequence of DNA from a microorganism that is under target. Subsequent to this, the sequence much be amplified in order to allow for detection of the microorganism. PCR is considered to be reliable and specific, as a detection method, being able to detect bacteria of pathogenic nature within a time frame of a day. As a form of DNA-based assay, PCR has been developed to detect foodbourne pathogens. For the purposes of DNA hybridization, PCR is able to amplify one single DNA copy in fewer than 2 hours by one million times. However, in situations where amplification is not completely efficient, such as when inhibitors are present in food, the normally extremely high levels of sensitivity of PCR become reduced. In order to improve sensitivity therefore, a form of cultural enrichment is likely to achieve this (Rose & Stringer, 1989).

As a rapid method to screen food samples for bacteria, PCR tests that are run and found to yield positive results are regarded as being ‘presumptive’ and require methods that are more conventional to confirm this (Feng, 1996). For direct testing, due to a lack of adequate specificity and sensitivity, pre-analsysis culture enrichment is frequently called for, which serves to increase specificity (Feng, 1997).

6.1 Sensitivity and specificity of PCR

To maximise the sensitivity of certain types of PCR, such as NK-1R PCR, a form of ‘nested’ PCR, and for this an increased number of cycles of the primary PCR may be helpful. For example, 35 secondary PCR cycles and 45 primary PCR cycles, were performed by O’Connell (2002) as opposed to a more standard number of between 25 and 30 cycles for both to increase sensitivity. In order to identify and detect bacteria furthermore, qcRT-PCR is likely to be less sensitive overall than more conventional PCR and hence, single-target PCR is advisable for a higher level of sensitivity.

It has also been noted that PCR conditions and parameters of cycling should ideally be optimised for every, and each primer in order to allow the achievement of a maximum yield of specific product and miminise monotarget sequence amplification. Knowles (1992) suggests that nested PCR may be helpful in improving both sensitivity and specificity. It is noted that increasing the speed of amplification of PCR has not effect upon test sensitivity, and hence this alteration it is unlikely to be worth the additional costs or time-saving advantage associated with increasing the cycling protocol.

7. Conclusion

Rapid tests such as PCR, the Enterotube II system, ELISA, flow cytometry and chromogenic methods have both benefits and limitations. The relative availability of these techniques and the speed of detection of bacterial pathogens, amongst other factors, suggest advantages but the sensitivity and specificity of the tests must be such that a reliable test result is ensured. In conclusion, a balance of sensitivity and specificity is required, but, by using the techniques mentioned, the reliability of the results obtained by the microbiologist is most likely to be improved.

Bibliography

Cantera, J.L., Chen, W., & Yates, M.V. 2010. Detection of Infective Poliovirus by a Simple, Rapid, and Sensitive Flow Cytometry Method Based on Fluorescence Resonance Energy Transfer Technology. Applied and Environmental Microbiology, 76(2), pp.584-588.

Cuzzubbo, A. J., Vaughn, D.W., Nisalak, A., Solomon, T., Kalayanarooj, S., Aaskov, J., Dung, N.M. & Devine, P.L. 1999. Comparison of PanBio Dengue Duo Enzyme-Linked Immunosorbent Assay (ELISA) and MRL Dengue Fever Virus Immunoglobulin M Capture ELISA for Diagnosis of Dengue Virus Infections in Southeast Asia. Clinical and Diagnostic Laboratory Immunology. 6(5), pp. 705-712.

Dalton, M.T., Comeau, S., Rainnie, B., Lambert, K & Forward, K.R.1993. A comparison of the API Uriscreen with the Vitek Urine Identification-3 and the leukocyte esterase or nitrite strip as a screening test for bacteriuria. Diagnostic Microbiology and Infectious Disease. 16(2), pp.93-97.

Downes, F.P. 2001. Compendium of methods for the microbiological examination of foods. 4th ed. Washington, DC: American Public Health Association.

Evans, D.J. Jr., Evans, D.G., Graham, D.Y. & Klein, P.D. 1989. A sensitive and specific serologic test for detection of Campylobacter pylori infection. Gastroenterology. 96(4), pp. 1004-1008.

Feng. P. 1996. Emergence of rapid methods for identifying microbial pathogens in foods. Journal of AOAC International. 79(3), pp.809-812.

Feng, P. 1997. Impact of Molecular Biology on the Detection of Foodborne Pathogens. Molecular Biotechnology. 7(3)., pp.267-278.

Gunasekera, T.S., Veal, D.A., & Attfield, P.V. 2003. Potential for broad applications of flow cytometry and fluorescence techniques in microbiological and somatic cell analyses of milk. International Journal of Food Microbiology, 85(3), pp.269-279.

Hoefel, D., Monis, P.T., Grooby, W.L., Andrews,S., & Saint, C.P. 2005. Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System. Applied and Environmental Microbiology. 71(11) pp. 6479-6488.

Knowles, D.M. (ed.). 1992. Neoplastic Hematopathology, 1st ed. Williams and Wilkins. pp. 919–930.

Monnery, I., Freydiere, A.M., Baron, C., Rousset, A.M., Tigaud, S., Boude-Chevalier, M., de Montclos, H. & Gille, Y. 1994. Evaluation of two new chromogenic media for detection of Salmonella in stools. European Journal of Clinical Microbiology and Infectious Diseases. 13(3), pp. 257-261.

Nolan, J.P & Sklar, L.A. 1998. The emergence of flow cytometry for sensitive, real-time measurements of molecular interactions. Nature Biotechnology, 16(7), pp. 633 – 638.

O’ Connell, J. 2002. RT-PCR Protocols. Totowa: Humana Press Inc.

O’Hara, C.M., 2005. Manual and Automated Instrumentation for Identification of Enterobacteriaceae and Other Aerobic Gram-Negative Bacilli. Clinical Microbiology Reviews. 18(1), pp. 147-162.

Painter, B.G. & Isenberg, H.D. 1973. Clinical laboratory experience with the improved Enterotube. Journal of Applied Microbiology, 25(6), pp. 896–899.

Perez, J.M., Cavalli, P., Roure, C., Renac, R., Gille, Y. & Freydiere, A.M. 2003. Comparison of four chromogenic media and Hektoen agar for detection and presumptive identification of Salmonella strains in human stools. Journal of Clinical Microbiology. 41(3), pp. 1130-1134.

Rose, S.A., & Stringer, M.F. 1989. Immunological methods, pp. 121-167. In: Rapid Methods in Food Microbiology: Progress in Industrial Microbiology. M.R. Adams and C.F.A. Hope (eds). New York: Elsevier.

Svennerholm, A., Lange, S. & Holmgren, J. 1878. Correlation between intestinal synthesis of specific immunoglobulin A and protection against experimental cholera in mice. Infection and Immunity. 21(1), pp. 1–6.

Woolfrey, B.F., Fox, J.M. & Quall, C.O. 1981. Evaluation of the Repliscan II System for identification of Enterobacteriaceae. Journal of Clinical Microbiology 14(4), pp. 408-410.


Categories
Free Essays

Identification of Gram negative bacteria using biochemical tests, including API

Abstract

Four pure, unidentified cultures of (gram positive cocci) bacteria, labelled A-D were cultured on various agar media. Also an API test was simulated to identify another unidentified bacterium. Identification of bacteria is important when choosing an effective treatment for a microbial-causing illness. This experiment focused on the cultural and biochemical characteristics of bacteria in aid of identification. Under aseptic conditions, each of the four unidentified bacterium were cultured using the bile aesculin, manitol salt and the blood agar plates provided. These were then incubated for over a week and then observed. A catalase and Voges-proskauer were also carried out to verify the identity of the 4 strains of bacteria. Bacteria that produced air bubbles in the catalase test (as oxygen is one of the products formed, in the presence of the enzyme catalase) and a red colour change for the Voges-proskauer (bacteria is able to produce a compound called acetylmethylcarbinol), both indicative of a positive result. For simplicity, the end cultures were compared with a table of results provided in the experiment to confirm the identity of Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus and Streptococcus pyogenes bacteria. The first culture easily identified as Streptococcus pyogenes produced a visible ?-haemolysis on blood agar; with an obvious clear zone around the colonies and was also unable to grow on manitol salt agar. The other strains were then determined from the various biochemical tests, as all bacterium possess particular characteristics that distinguish them from other genera. The bacterium used in the API was identified as Staphlyococcus. aureus, by use of an identification table, provided by the manufacturer of the API. However in a normal setting various other tests would have to be conducted to conclude the genus and species of the bacteria.

Introduction

Gram positive and gram negative bacteria have a rigid cell wall called peptidoglycan and this can be used to distinguish between the two groups. Gram positive bacteria have a very thick outer layer of peptidoglycan. They also have the lipopolysaccharide layer absent. (Madigan et al., 2009) Gram positive bacteria usually appear purple and gram negative bacteria can be red to pink in colour with the use of gram staining. (Madigan et al., 2009)

Once established the fact that the bacterium belong to gram positive group, the Dichotomous Key of Gram Positive bacteria can be used to differentiate bacteria by use of various biochemical tests. (Willey et al. 2008)

The isolation and identification of bacteria is an essential diagnostic tool in microbiology, especially investigating pathogenic bacteria that cause infectious diseases. The clinician and microbiologist work together in this identification process. (Willey et al. 2008) Samples from the suspected infected area of a patient can be extracted and grown aseptically on agar medium to avoid contamination; these mixed cultures are then separated to produce single colonies of a genus bacterium. The shape of the bacteria can be determined by microscopy (using gram staining or other staining techniques for acid-fast bacteria), and culturing of the bacteria on various media – selective, differential and certain characteristic (metabolic) media. (Willey et al. 2008) Selective media only allow certain bacteria to grow, whilst differential media are used to distinguish bacteria from others, in the presence of some form of dye or indicator. (Madigan et al., 2009) It is also important to note the conditions bacteria are able to grow in, as some may tolerate the presence of oxygen (aerobes) whilst others will not (anaerobes). The presence of specific enzymes enables aerobic bacteria to grow, whilst anaerobic bacteria cannot. (Madigan et al., 2009) Voges-Proskauer tests distinguish bacteria that are able to produce fermentation, especially when they cannot respire aerobically. (Willey et al. 2008)

When microscopy and culturing methods alone are not adequate enough to identify a species, specific biochemical tests are carried out. These tests are used to eliminate the number of possible pathogens causing the illness in question; by comparing the unidentified pathogen with the known metabolic characteristics stored on computer databases. (Madigan et al., 2009) These may include testing for products the bacterium may produce (due to a presence of specific enzyme/s) or even their ability to grow on either selective or differential media or a combination of the two. However some require further investigative tests to identify the bacteria. (Madigan et al., 2009)An example is the coagulase test, which differentiates S.aureus from S.epidermidis, coagulase has the ability to clot plasma. (Willey et al. 2008) Once the bacteria have been identified, antibiotic sensitivity tests (susceptibility tests) may be performed in order to determine which antibiotic/s would be most effective in treating the illness related to the microorganism. (Willey et al. 2008)

The ability of bacteria to produce catalase is an important biochemical characteristic, aerobic bacteria are able to secrete specific enzymes this characteristic can be manipulated in identification. (Madigan et al., 2009) Aerobic bacteria are able to neutralise hydrogen peroxide (that would otherwise be toxic to it) by converting it to water and oxygen. Bubble formation would indicate a positive result of this reaction taking place. (Greenwood et al., 2007) This test helps to identify streptococcus from staphylococcus. (Willey et al. 2008) Further more some bacteria may have the ability of secreting other enzymes like superoxide dismutase and peroxidise. This depends on the growth conditions the bacteria require, to neutralise free (unpaired) oxygen radicals that would otherwise destroy the normal functioning of bacterial cells. These radicals are the result of oxygen being reduced in the electron transport chain. (Willey et al., 2008)

Indicator medium of blood agar (usually containing horse blood) is used for the haemolysis test to indicate if the bacterium produces a specific toxin (haemolysin) this is a common virulence factor that pathogenic bacteria possess. A positive result indicates the bacterium possesses this toxin. (Willey et al. 2008) The toxin is able to lyse erythrocytes by forming pores in the cell surface, releasing its contents – haemoglobin and other ions. (Willey et al., 2008) This can be observed on blood-agar as a clear halo with no distinct colour around the colonies, called ?-haemolysis. Partial (?) haemolysis leaves a slight green discolouration, as hydrogen peroxide oxidises haemoglobin to methaemoglobin. (Greenwood et al., 2007)

Bile aesculin agar is selective and differential, black formation on the culture plate would indicate the ability of the bacterium to hydrolyse aesculin and mix with ferric citrate. (Mahon and Manuselis, 2000) The manitol salt agar is an example of selective media that only allows growth of specific bacteria to grow, thus it can be used in biochemical tests. This is due to the high concentration of salt within this medium, which inhibits most bacteria from growing. (Mahon and Manuselis, 2000)

Rapid identification of a microorganism can be determined by the use of an API (Analytical Profile Index) or manual ‘kit’ (Willey et al. 2008)that contains 20 microtubules with dehydrated substrates, once inoculated with bacteria and left to incubate; the various wells produce colour changes when reagents are added. These colour changes are related to the metabolic characteristics of specific bacteria that can be matched to an identification table.

The use of current technology enables one to study the genomic and antigenic structure of microorganisms and is thus useful in identification. The use of PCR and electrophoresis can be used in Multilocus Sequence Typing (MLST) and genomic fingerprinting. (Willey et al. 2008)Also the various surface proteins especially antigens can be identified for its interaction with particular antibodies by immunofluorescence or agglutination technique. This technique may yield rapid results and streptococci associated with sore throats can be identified this way; however these tests are not as accurate as the culturing techniques. (Champoux et al., 2004) New and more accurate technologies are being studied such as the use of Biosensors. (Willey et al., 2008)

Staphylococci have a round shape (from the Greek word ‘kokkos’ meaning a berry.) these bacteria form clusters like grapes (derived from the Greek word ‘staphule’) Staphylococci also have a slime layer, and are mainly found on the surface of skin.(Heritage et al., 1999) These aerotolerant anaerobe are able to grow in either aerobic or anaerobic conditions. Although Staphylococcus aureus is harmless living on the surface of the skin, it is able to cause serious illness like septicaemia when it enters open wounds. (Mandal et al., 1996) This bacterium can also become an opportunistic pathogen, responsible for epidemics like MRSA due to resistance of the antibiotic methicillin and emerging resistance to vancomycin. (Willey et al., 2008) A quick biochemical test called Staphaurex can also be used. (Willey et al., 2008)

Streptococci are facultative anaerobes and do not form any gas products, as they produce lactic acid fermentation and will therefore catalase negative. (Willey et al., 2008) The streptococcus genera cover an extensive group of bacteria – the cocci that are spherical in shape and thus placed into 3 groups: pyogenic, oral and other (colon) streptococci. (Greenwood et al., 2007) Virulence factors produced by the pathogenic bacteria (pyogenic) like the presence of streptolysin, have the ability to lyse erythrocytes and can inhibit the host’s immune response as it kills leukocytes. Haemolysis is a key step to identify pyogenic (harmful) streptococci from other streptococci. (Willey et al., 2008)

The species E. faecalis can be found in the intestinal tract, it has the ability to cause opportunistic infections like urinary tract infections (UTI) and also is able to grow in 6.5% sodium chloride, and can resist certain antibiotics. (Willey et al., 2008) The enterococcus group are closely related to the streptococcus group, but are associated more within the intestinal area. (Champoux et al., 2004) The species M.luteus are obligate aerobes in that they rely completely on oxygen to survive and so can be found on one’s own microbiota, the surface of skin. (Madigan et al., 2009)

Method

A week before identification, 4 unidentified pure strains labelled (A-D) were each cultured on blood, bile aesculin and manitol salt agar that corresponded to each letter. The streak-plate technique was applied, a loop used to transfer the bacteria to the agar plates was sterilised under an open flame and left to cool, before each set of streaks. After a week, the agar plates were all examined and the type of results they produced was recorded. A single colony (seen by naked eye) was removed from the original (ordinary) agar plates. Each of these was inoculated over a few days and used for the Voges-Proskauer test. The reagents alpha napthol and 40% KOH were added, the tubes were then observed for colour changes. Also a catalase test was carried out, an inoculated loop was used to transfer a small amount from each strain (from the ordinary agar plates) to a microscope slide and hydrogen peroxide was added. Those that bubbled were noted as positive. All results from the various biochemical tests were compiled in table format – the catalase; Voges-Proskauer; haemolysis (blood agar); ability to produce aesculetin (bile agar) and ability to grow (on manitol salt agar). The 4 strains of bacteria were thus identified.

Separately, an API test was simulated of an ‘unidentified’ staphylococci bacterium. Each well of the incubation box for the API was filled with distilled water followed by an ampoule of the bacteria which was inoculated and prepared to the correct McFarland standard tube of 0.5. Mineral oil filled the outlined wells. The box was incubated for a few days; reagents were added to the corresponding wells and after 10 minutes observed for colour changes. Reagents VP1 and VP2 were added to the VP well; NIT1 and NIT2 to NIT well and lastly Zym A and Zym B to PAL well. The test colour result for each well was then noted (either positive or negative) on an API Staph strip and matched with the identification table of the various Staphylococcus species. The staphylococcus species was thus identified.

Results

The 4 unidentified strains (labelled A-D) were exposed to various biochemical tests, the results from these are given below.

Table 1: Results from the gram positive strains

Results from the API test:

A bacterium was then identified by the use of API test, a colour indication table was also provided to determine if the results were positive or negative. These results were jotted down on a test strip and compared with a test table to identify the species of Staphyloccocus.

Figure 1: Test strip

Figure 2: Identification Table of Staphylococcus species (Provided by API Manufacturer)

Discussion

Observation of the colour and characteristics of the pathogen, with the use of various biochemical tests can identify the bacterium causing the infection. (Madigan et al., 2009) This can be applied in this experiment.

Referring to Table 1: The ability to produce haemolysis is dependent on bacteria to secrete a toxic substance called haemolysin, which is able to lyse red bloods cells. Thus blood agar is used which is a differential medium. (Willey et al., 2008) The type of haemolysis bacteria produce can be observed by the naked eye, as clearing zones around the colonies. A ?-haemolysis results in distinct, colourless clear zones of colonies, as the erythrocytes (of the blood agar) have completely lysed. The species S. pyogenes has the ability to secrete exotoxins, depending incubation conditions it will either secrete Streptolysin-O (anaerobic) or Streptolysin-S (aerobic). The pyogenic bacteria are distinguished from other streptococci by producing ?- haemolysis. (Willey et al., 2008) Whilst ?-haemolysis is the partial destruction of erythrocytes with some clearing and slight green discolouration, it is not as distinct as ?-haemolysis. The green tinge is a result of haemoglobulin being oxidised. Conversely M luteus and E. Faecalis produce ?-haemolysis i.e. no colour change or clearing zone on the agar as the bacteria are unable to produce haemolysin. (Greenwood et al., 2007)

Furthermore the Lancefield method together with haemolysis testing can be used to identify pathogenic streptococci from other less evasive streptococci. (Greenwood et al., 2007) The Lancefield method involves the agglutination of antibodies with the cell wall antigens (C polysaccharide) each serotype is classified A-T, depending on the sort of antigen-polysaccharide nature of this reaction. (Willey et al., 2008).

Voges-proskauer is used to indicate if the bacteria in question produce fermentation, this would depend on their culture needs – especially anaerobic bacteria which are unable to respire without the electron transport chain. (Willey et al., 2008) The red colour produced is a test positive for the production of acetoin or acetylmethylcarbinol in glucose fermentation. (Champoux et al.,2004) Referring to Table 1, S. aureus tests positive as it’s a facultative anaerobe. (Willey et al., 2008) Whilst M. luteus and Str. pyogenes can grow in aerobic conditions and so do not require the principles of fermentation, they test negative.

Conversely, unlike anaerobes ability to produce fermentation, most aerobes possess the enzyme catalase. A positive catalase test results in bubble formation when hydrogen peroxide is added to a bacterium. The enzyme catalase is able to form water and oxygen from hydrogen peroxide. (Madigan et al., 2009) The Streptoccocus pyogenes produce no gas, and instead utilise lactic acid to break down sugars. (Willey et al., 2008) They catalase negative as the enzyme catalase is not present; so cannot break down hydrogen peroxide to form water and oxygen. (Greenwood et al., 2007) Staphylococcus tests positive and can utilise glucose to form acidic products. (Madigan et al., 2009) It’s also an aerotolerant anaerobe, it may lack the enzyme superoxide dismutase which can break down superoxide radicals, but can make use of manganese ions instead. This may have been an adaptative mechanism when the very first forms of bacteria were exposed to oxygen. (Madigan et al., 2009) This enzyme is common in most pathogenic bacteria, and increases their virulence by neutralising the otherwise toxic hydrogen peroxide and minimizing death by phagocytosis by host cells. (Champoux et al., 2004) M. luteus grow in aerobic conditions and can only utilise glucose in these conditions, this would explain why it would catalase positive, to neutralise toxic hydrogen peroxide. (Madigan et al., 2009)

Bile aesculin agar is selective and differential, black formation on the culture plate would indicate the ability of the bacterium to hydrolyse aesculin and mix with ferric citrate. (Mahon and Manuselis, 2000) The presence of bile salts will inhibit some types of bacteria like S. pyogenes and M.luteus (as seen on Table 1)

Manitol is a selective media, only allowing some bacteria to tolerate it, like S. aureus and E. faecalis. They are both able to utilise manitol by fermenting it to produce acid, thus lowering the pH the agar changes from red to a yellow colour as a result. Incubation of S. aureus is slightly longer, and so a coagulase test can also be implemented. (Mahon and Manuselis, 2000) Whilst haemolysis identifies pathogenic streptococci like Str. pyogenes, the manitol agar identifies pathogenic staphylococci. Also Str. pyogenes cannot grow on this agar, and so no visible colonies are formed. (Willey et al., 2008) While M. luteus has the ability to grow on manitol salt agar (visible colonies), so one would assume that it cannot utilise manitol, as there is no colour change present as it cannot produce acid.

As mentioned, there are various API tests available; this experiment used an API Staph Test which identified Staphylococcus, micrococcus and kocuria genera. (CITATION) The test kit was compared with the colour change table of the various substrates (when reagents are added) and the API test strip was marked accordingly for a positive or negative result. The test strip (Figure 1) was then compared to the identification table (Figure 2) and the unknown bacterium was identified as S. aureus. The limitations of this test is that a pure culture of bacteria must be used and that API’s are specific for a particular genera of bacteria, various API tests are available (http://www.biotech.ug.edu.pl/odl/biochem/api.html) these include an API 20E to identify Enterobacteriaceae (Willey et al., 2008) Also any experimental error like not adding reagents correctly to specific well can also give false positives, thus not correctly identifying the species.

Conclusion

Identification of gram positive bacteria can be achieved by carrying out various biochemical tests. Differential media like blood agar is useful in identifying the type of haemolysis and thus the pathogenicity of various bacteria (streptococci). Selective media like manitol salt agar inhibits growth of certain bacteria like streptococci, whilst also determining the presence of particular enzymes by the end products produced, this can be observed by colour changes. Various other biochemical tests are available and can produce rapid results – like the API. The simulation of identifying bacteria in this experiment, accentuated how vital these tests are in order to treat patients effectively. However it should be noted in realistic settings further biochemical tests and the use of modern technologies may be required to correctly identify microorganisms.

References
Greenwood, D.; Slack, R.; Peutherer, J., Barer, M. (2007) Medical Microbiology A Guide to Microbial Infections: Pathogenesis, Immunity, Laboratory Diagnosis and Control. 17th ed. Philadelphia: Elsevier Limited.
Heritage, J., Evans, E.G.V., Killington, R.A. (1999). Introductory Microbiology. Cambridge: The Press Syndicate of The University of Cambridge
Madigan, M., Martinko, J., Dunlap, P., Clark, D,. (2009). Brock Biology of Microorganisms. 12th ed. San Francisco: Pearson Benjamin Cummings
Mahon, C.R., Manuselis, G. (2000) Textbook of Diagnostic Microbiology. 2nd ed. Philadelphia: Saunders An Imprint of Elsevier
Mandal, B.K.; Wilkins, G.L.E.; Dunbar, E.M.; Mayon-White, R.T. (1996) Infectious Diseases. 5th ed. Oxford: Blackwell Science Ltd.
Champoux, J.J., Drew, W.L., Neidhardt, F.C., Plorde, J.J.(2004) Sherris Medical Microbiology. 4th ed. USA: McGraw-Hill Companies, Inc.
Willey, J.; Sherwood, L., Woolverton, C. (2008) Prescott, Harley, and Klein’s Microbiology. 7th ed. New York: McGraw-Hill.

Categories
Free Essays

“Rapid detection and identification of bacteria in food and clinical laboratories”

Abstract

Modern technological progress has affected how microbiology is practiced. There is emphasis on the minimalisation of laboratory costs, cost-efficiency and reliability of tests for efficient bacterial identification from food cultures. Before using any technology, it is recommended that the products’ performance characteristics be first tested, particularly as theses characteristics, are often not determined by the manufacturers. Consequently, the sensitivity and specificity, amongst other factors, associated with the use of these tests will also not have been determined. Additional factors would benefit from the use of controls, such as in the form of large scale and controlled clinical trials, in order to study the products’ performance. It is to be borne in mind that the involvement of ‘rapid’ tests, including an enzyme-linked immunosorbent assay, in bacterial detection may serve bests as methods for expeditious detection and screening than for the purposes of confirmation.

Keywords: ELISA, flow cytometry, FCM, enterotube ll system, Chromogenic media, PCR.

___________________________________________________________________

1. Introduction

In order to help diagnose infectious diseases, such as the bacteria Salmonella, a leading cause of food poisoning, the need for specialised microbial tests has arisen. Testing food products using rapid methods is a complicated process requiring the balance of sensitivity and specificity for the achievement of a reliable result. The following sections will discuss the use of five different detection methods, flow cytometry, the enterotube II system, chromogenic media, the Enzyme linked immunoassay and polymerase chain reaction and the necessity to balance the specificity and sensitivity of each technique, for the most accurate means of bacterial detection.

2.0 Flow Cytometry

Flow cytometry (FCM) is based on the principles of excitation of light, light scattering and fluorochrome molecular emission for the purposes of generating data covering a number of different parametric readings. FCM focuses on cells that measure 0.5um to 40 ?m in diameter. The technique of FCM relies on the provision of a light source, which, are usually lasers, and the cells must first be covered in a layer of phosphate buffered saline before being able to intercept the focused source of light. In this technique, a sample, containing the cells being tested, are injected into the centre of a sheath flow. Flow cytometry provides an analysis of cellular interactions at the macromolecular level. FCM is a technique that is considered to be a critical component of research in the biomedical field (Nolan & Sklar,1998).

2.1 Milk testing

FCM is one technique which may be useful when testing the safety and quality of milk. Testing milk requires analysis of somatic cell count and microbial analysis. Tests have shown (Gunasekera, et al., 2003) that the analysis of milk, where a known number of cells have been inoculated, upon clearing can be performed by FCM. FCM is able to give a good indication of the somatic cell count in raw milk and when coupled with other methods such as techniques involving fluorescence staining, can be used in testing biological milk quality. This therefore has an important application in the dairy industry, particularly in quality testing.

2.2 Analysis of Water Quality

The use of flow cytometry has to date also occurred in tandem with heterotrophic plate count (HPC) for the rapid detection of the bacterial count of potable as well as raw water (Hoefel, et al., 2005). The results showed that FCM was much quicker than HCP, in detecting viable bacteria in samples that were classed as viable but not amenable to culture. The FCM method detected bacteria within an hour as opposed to several days, for the HCP technique.

Studies have tested the sensitivity of FC-based assays in comparison to the plaque assay method, to measure levels of an infection virus in a sample (Cantera, et al., 2010). Poliovirus infection (PV1) was tested and the FCM method applied to a water sample infected with PV1-infected cells. The study revealed that a combination of flow cytometry, used with fluorescence resonance energy transfer technology, is able to sensitively and quickly detect the presence of infectious virus in a sample of environmental water.

2.3 Specificity of FCM

FCM has also been used to investigate whether T4 phage infected cells with E. coli ATCC 111303 can be differentiated from uninfected cells, based on phage DNA fluorescent detection. The technique, involving the lysis of bacterial cells by phage, allowed for the detection for infected cells 35 minutes post infection. Thus, FCM is able to be specific, when used combined with phages of predetermined host specificity. Overall, FCM is able to quantitatively measure and sensitively detect molecular level interactions and as such it may be considered to be a robust and adaptable technology (Nolan & Sklar, 1998).

3.0 The enterotube ll system

The Enterotube II was described for the first time in 1969 (Painter & Isenberg, 1973). This technology is an example of a rapid system of multi-test nature, functioning as a biochemical and enzymatic test method. The test system, functions by identifying unclassified gram-negative, rod shaped and oxidase-negative bacteria, belonging to the family Enterobacteriaceae. The test is often conducted within clinical laboratories. The machine comprises a flat-sided tube within which are 12 compartments, developed to allow different biochemical tests to be conducted. The system does consistently produce accurate results, and hence is liable to produce occasional false results.

3.1 Sensitivity and specificity

Reports such as the one by Dalton et al., (1993) in the detection of bacteriuria, have found that upon screening, only 55% specificity and 93% sensitivity have been obtained. O’Hara (2005) reports that it may be valuable for the diagnostic laboratory running tests, using equipment such as the Enterotube II system, to first stipulate what levels of ‘accuracy’ and ‘discrimination’ they consider are acceptable from their systems of identification. Accuracy of identification may be maximised by using the skills of a qualified microbiologist to confirm the bacterial classification (O’Hara, 2005). An additional way to potentially maximise sensitivity and specificity is to send an isolate to a reference laboratory in order to confirm identity. Use of enterotube II system will be for the testing of oxidase-negative bacteria and hence it should first be established that the oxidase test is not positive. To achieve this, and improve the specificity, an oxidase test may be performed on the relevant cultures.

In order to improve interpretation of results from use of the Enterotube II system, a suitable incubation time should be used, such as 16 hours (in the analysis of carbohydrate reactions (Woolfrey, et al., 1981). Furthermore, tests resulting in ambiguous classifications should be reevaluated (Woolfrey, et al., 1981) in order to improve specificity, without hampering the tests’ sensitivity.

4.0 Chromogenic Media

Chromogenic media (or fluorogenic media) are a microbial growth media of microbial nature. The media contains enzymes that are linked to either fluorogen (involved in light reaction) or chromogen (involved in colour reaction) or a combination of both. The method works by detecting activities that are enzymatic in nature, that are produced by the target microorganisms. Enzymatic activities are detected by the use of either organic compounds or dyes, as microorganisms, which grow in the proximity of these compounds are liable to make a distinctive pattern of colouring or alternatively fluoresce, which can be detected under UV light. Chromogenic media were first designed for application in clinical settings, but have proven to be useful in food testing.

4.1 Sensitivity and specificity of chromogenic media

Chromogenic media are considered to be a sensitive method of media analysis, when compared to more conventional types of media analysis (Downes, 2001). This is because the chromogenic media method allows for a faster analysis, with a turnover time of 24 hours, and it is also considered to have a higher sensitivity. In the identification of E.coli or Listeria monocytogenes, for example, specially designed chromogenic media are available for the purposes of improving test sensitivity.

When considering Salmonella detection, a number of specialised chromogenic media that are able to improve the specificity of detection are available. A study by Perez et al., (2003) showed that both broth enrichment and increasing the incubation time by a factor of two (from 24 hours to 48 hours) effectively increases the sensitivity of all of the media being used. Furthermore, due to the specificity of the chromogenic media, (determined to be greater than 84% following a two-day incubation period), a reduction in the need to undergo confirmatory tests improved the overall sensitivity of the specialized chromogenic media. A second study by Monneri et al., (1994), for the comparison of two new types of agar, media of chromogenic nature, Salmonella Detection and Identification Medium (SMID) and Rambach agar, against two conventional types of media for the detection of Salmonella. The results revealed that the newer chromogenic agar media were notably more specific than the more conventional media. Rambach agar was furthermore slightly more specific than SMID, being able to detect all Salmonella serotypes following a complementary C8 esterase test. Hence, sensitivity and specificity can be maximised by increasing culture time to 2 days fully, and using Rambach agar where appropriate, such as in the detection of Salmonella serotypes.

5.0 Enzyme Linked Immunoassay

The Enzyme linked immunoassay (ELISA) is a common antibody based technique designed for microorganism, or pathogenic, detection. The method is noted to have a high standard of specificity and sensitivity (Evans et al., 1989). A quantitative, or qualitative method may be used for the purposes of interpreting the results, which are, respectively, via the use of an instrumental read-out or through visual means. Specialised test kits to aid in the detection of Listeria, Salmonella and other microorganisms are commercially available.

5.1 Sensitivity and specificity

A study by Evans et al., (1989) utilised ELISA in the detection of Campylobacter pylori. The specificity and sensitivity of the test allowed for the detection of serum immunoglobulin G (IgG) antibodies targeted against the cell-associated proteins of C. pylori. Values of specificity and positive predictive value were revealed to be 100% for the high molecular weight cell-associated proteins. Furthermore, the assay sensitivity was measured at 98.7%, with the negative predictive value recorded as 98.6%. This indicates that specialised ELISA tests are likely to be valuable in such instances as in the detection of H. pylori. Furthermore, the costs of using the ELISA, as noted by Evans et al., (1989) are that it is cost effective and readily usable, with a lower likelihood of obtaining false negatives than with other tests, such as the use of a ‘urea breath test’ which is also amenable to be useful for the same purpose.

Svennerholm & Holmgren, (1978) report that E. Coli can be sensitively detected using a ganglioside ELISA. The method was deemed to be reliable and allow a high level of reproducibility. In general, it has been reported that the specificity and, or, sensitivity of assays that are commercially available, such as the ELISA may be maximised by having set cut-off values decreed by the manufacturers, according to the target disease (Cuzzubbo, et al., 1999). Furthermore, the IgG test, due to having 100% specificity, is highly likely to be reliable, as a method for bacterial testing.

6.0 Polymerase Chain Reaction

Similar to the ELISA test, the ‘PCR’ or the polymerase chain reaction (PCR) is one of the most readily recognised and used diagnostic tool currently in use. PCR works by identifying a highly specific sequence of DNA from a microorganism that is under target. Subsequent to this, the sequence much be amplified in order to allow for detection of the microorganism. PCR is considered to be reliable and specific, as a detection method, being able to detect bacteria of pathogenic nature within a time frame of a day. As a form of DNA-based assay, PCR has been developed to detect foodbourne pathogens. For the purposes of DNA hybridization, PCR is able to amplify one single DNA copy in fewer than 2 hours by one million times. However, in situations where amplification is not completely efficient, such as when inhibitors are present in food, the normally extremely high levels of sensitivity of PCR become reduced. In order to improve sensitivity therefore, a form of cultural enrichment is likely to achieve this (Rose & Stringer, 1989).

As a rapid method to screen food samples for bacteria, PCR tests that are run and found to yield positive results are regarded as being ‘presumptive’ and require methods that are more conventional to confirm this (Feng, 1996). For direct testing, due to a lack of adequate specificity and sensitivity, pre-analsysis culture enrichment is frequently called for, which serves to increase specificity (Feng, 1997).

6.1 Sensitivity and specificity of PCR

To maximise the sensitivity of certain types of PCR, such as NK-1R PCR, a form of ‘nested’ PCR, and for this an increased number of cycles of the primary PCR may be helpful. For example, 35 secondary PCR cycles and 45 primary PCR cycles, were performed by O’Connell (2002) as opposed to a more standard number of between 25 and 30 cycles for both to increase sensitivity. In order to identify and detect bacteria furthermore, qcRT-PCR is likely to be less sensitive overall than more conventional PCR and hence, single-target PCR is advisable for a higher level of sensitivity.

It has also been noted that PCR conditions and parameters of cycling should ideally be optimised for every, and each primer in order to allow the achievement of a maximum yield of specific product and miminise monotarget sequence amplification. Knowles (1992) suggests that nested PCR may be helpful in improving both sensitivity and specificity. It is noted that increasing the speed of amplification of PCR has not effect upon test sensitivity, and hence this alteration it is unlikely to be worth the additional costs or time-saving advantage associated with increasing the cycling protocol.

Conclusion

Rapid tests such as PCR, the Enterotube II system, ELISA, flow cytometry and chromogenic methods have both benefits and limitations. The relative availability of these techniques and the speed of detection of bacterial pathogens, amongst other factors, suggest advantages but the sensitivity and specificity of the tests must be such that a reliable test result is ensured. In conclusion, a balance of sensitivity and specificity is required, but, by using the techniques mentioned, the reliability of the results obtained by the microbiologist is most likely to be improved.

Bibliography

Cantera, J.L., Chen, W., & Yates, M.V. 2010. Detection of Infective Poliovirus by a Simple, Rapid, and Sensitive Flow Cytometry Method Based on Fluorescence Resonance Energy Transfer Technology. Applied and Environmental Microbiology, 76(2), pp.584-588.

Cuzzubbo, A. J., Vaughn, D.W., Nisalak, A., Solomon, T., Kalayanarooj, S., Aaskov, J., Dung, N.M. & Devine, P.L. 1999. Comparison of PanBio Dengue Duo Enzyme-Linked Immunosorbent Assay (ELISA) and MRL Dengue Fever Virus Immunoglobulin M Capture ELISA for Diagnosis of Dengue Virus Infections in Southeast Asia. Clinical and Diagnostic Laboratory Immunology. 6(5), pp. 705-712.

Dalton, M.T., Comeau, S., Rainnie, B., Lambert, K & Forward, K.R.1993. A comparison of the API Uriscreen with the Vitek Urine Identification-3 and the leukocyte esterase or nitrite strip as a screening test for bacteriuria. Diagnostic Microbiology and Infectious Disease. 16(2), pp.93-97.

Downes, F.P. 2001. Compendium of methods for the microbiological examination of foods. 4th ed. Washington, DC: American Public Health Association.

Evans, D.J. Jr., Evans, D.G., Graham, D.Y. & Klein, P.D. 1989. A sensitive and specific serologic test for detection of Campylobacter pylori infection. Gastroenterology. 96(4), pp. 1004-1008.

Feng. P. 1996. Emergence of rapid methods for identifying microbial pathogens in foods. Journal of AOAC International. 79(3), pp.809-812.

Feng, P. 1997. Impact of Molecular Biology on the Detection of Foodborne Pathogens. Molecular Biotechnology. 7(3)., pp.267-278.

Gunasekera, T.S., Veal, D.A., & Attfield, P.V. 2003. Potential for broad applications of flow cytometry and fluorescence techniques in microbiological and somatic cell analyses of milk. International Journal of Food Microbiology, 85(3), pp.269-279.

Hoefel, D., Monis, P.T., Grooby, W.L., Andrews, S., & Saint, C.P. 2005. Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System. Applied and Environmental Microbiology. 71(11) pp. 6479-6488.

Knowles, D.M. (ed.). 1992. Neoplastic Hematopathology, 1st ed. Williams and Wilkins. pp. 919–930.

Monnery, I., Freydiere, A.M., Baron, C., Rousset, A.M., Tigaud, S., Boude-Chevalier, M., de Montclos, H. & Gille, Y. 1994. Evaluation of two new chromogenic media for detection of Salmonella in stools. European Journal of Clinical Microbiology and Infectious Diseases. 13(3), pp. 257-261.

Nolan, J.P & Sklar, L.A. 1998. The emergence of flow cytometry for sensitive, real-time measurements of molecular interactions. Nature Biotechnology, 16(7), pp. 633 – 638.

O’ Connell, J. 2002. RT-PCR Protocols. Totowa: Humana Press Inc.

O’Hara, C.M., 2005. Manual and Automated Instrumentation for Identification of Enterobacteriaceae and Other Aerobic Gram-Negative Bacilli. Clinical Microbiology Reviews. 18(1), pp. 147-162.

Painter, B.G. & Isenberg, H.D. 1973. Clinical laboratory experience with the improved Enterotube. Journal of Applied Microbiology, 25(6), pp. 896–899.

Perez, J.M., Cavalli, P., Roure, C., Renac, R., Gille, Y. & Freydiere, A.M. 2003. Comparison of four chromogenic media and Hektoen agar for detection and presumptive identification of Salmonella strains in human stools. Journal of Clinical Microbiology. 41(3), pp. 1130-1134.

Rose, S.A., & Stringer, M.F. 1989. Immunological methods, pp. 121-167. In: Rapid Methods in Food Microbiology: Progress in Industrial Microbiology. M.R. Adams and C.F.A. Hope (eds). New York: Elsevier.

Svennerholm, A., Lange, S. & Holmgren, J. 1878. Correlation between intestinal synthesis of specific immunoglobulin A and protection against experimental cholera in mice. Infection and Immunity. 21(1), pp. 1–6.

Woolfrey, B.F., Fox, J.M. & Quall, C.O. 1981. Evaluation of the Repliscan II System for identification of Enterobacteriaceae. Journal of Clinical Microbiology 14(4), pp. 408-410.

Categories
Free Essays

Isolation of Bacteria

Different types of bacteria in various forms are found all around us, and it is a microbiologist’s job to be able to identify these bacteria. Using various staining techniques and physiological tests, an isolated bacterium can be identified. In this experiment, a single bacterial colony was isolated form Mycorrhizal spores, and further tests done on that colony.

Sub culturing was done after each week to ensure that the bacterium has sufficient nutrients required for optimum growth that will last the duration of the entire experiment. A flow chart was created based on the results of the physiological tests in order to identify the isolated bacterium. After 4 weeks, the isolated bacterium was identified as XXXXX for reasons stated in the results and discussion.

The main goal of this experiment was to identify the isolated bacterium that was obtained from Mycorrhizal spores. In order to identify the bacterium, the experiment was conducted in 4 parts: (a) isolation of an unknown bacterium from soil; (b) identification of the bacterium using various staining techniques; (c) determining the motility of the bacterium; and (d) determining the physiological characteristics of the bacterium. Part (a) of the experiment involves isolating a single bacterial colony from the culture.

The remaining 3 parts will be conducted on that colony. In part (b), it is shown that various staining techniques test for different characteristics. As the name suggests, a gram stain is conducted to identify the bacteria as gram negative or gram positive. Two other stains were carried out. To determine the motility of the bacterium, wet mounts of the bacterium were observed and the motility was confirmed by using soft agar plates and soft agar deeps for part (c).

The physiological characteristics were identified in part (d). Some of these tests include growth temperatures and salt tolerance, degradation of polysaccharides, proteins and lipids, oxygen requirements etc. Based on the results for the above, the unknown bacterium can be identified by comparing it to cultures in the Bergey’s manual. A flow chart can be drawn up to correctly identify the bacterium by using the physiological test results.

Categories
Free Essays

Bacteria Transformation in Biotechnology

Abstract Some bacteria are able to go through transformation making new combinations of genes. Transformation is a way of gene variability in bacteria. This experiment is based on the transformation mechanism of bacteria and gene regulation. The bacteria used for the experiment was Escherichia coli and the genes introduces for the transformation were: gfp and bla by a pGLO™ plasmid. After the insertion of the target genes and growing the bacteria on specialized LB media, it could be seen that the transformants were positive for the gene expression.

The transformed E. coli on the media appeared fluorescent green under UV light. Introduction The bacteria used in this experiment is Escherichia coli which is not naturally competent. E. coli is a gram negative rod shaped bacteria and a facultative anaerobe. This bacteria forms part of the bacterial flora in the human intestine tract. The competence of a bacteria is based on its ability to take up naked DNA from the environment and incorporated on theirs, transformation. Alteration in the permeability of the membranes allows DNA to cross the cell envelope of E. oli. Since the outer membrane of the E. coli is mostly negatively charged and the DNA molecule also has a negative charge, then the addition of CaCl2 will neutralize the interaction so that the naked DNA molecule can enter the cell. (Microbe Library web) Another important factor on the competence of the bacteria is a procedure of alternating temperature between ice bucket and heat shocks. By the combination of this two procedures E. coli becomes competent. This procedure was first reported by Mandel and Higa. Singh 562) Even though it works it is only believed that CaCl2 helps DNA absorption to cell surface and the heat-shock step facilitates penetration of absorbed DNA into cell. (Panja 411) The main purpose of this experiment is to transform the bacteria to make it resistant to the antibiotic ampicillin. A secondary transformation is being made, and is to make the bacteria seem fluoresce green. The reason why the bacteria will fluoresce is because the gfp gene is being inserted under an ara promoter. The gfp gene encodes for the Green Fluorescent Protein (GFP).

The genes under the ara promoter will be expressed when the bacteria is in presence of the sugar Arabinose. When the transformed E. coli is in presence of Arabinose, the gfp will make the GFP and when the bacteria is placed under UV light it will fluoresce green. The gfp gene was found and extracted from a jellyfish, Aequorea victoria, and is being used as a visible reporter for gene expression. (Garcia-Cayuela 172) To introduce the gfp into the bacterial cell it was needed to be by a plasmid, as well as the gene to make the E. oli resistant to ampicillin, bla gene. The bla gene encodes for the protein beta lactamase which breaks down the ? -lactam ring in the structure of the ampicillin, therefore making it resistant to the antibiotic. Like already said to introduce this two genes to the E. coli it must be done through a plasmid. Both genes were introduced by the same one. In this case the one that was used was a pGLO™ plasmid. This is an engineered plasmid used as a vector to create genetically modified bacteria. This plasmid contains three specific genes: bla, gfp and araC.

The ¬araC is a promoter region that regulates the expression of the gfp only under the presence of arabinose sugar. Materials and Methods In this experiment a pGLO™ transformation kit was used. First we needed two eppie tubes, one pGLO positive and the other pGLO negative. This two eppies were then moved to an ice bucket. During, one loopful of the pGLO plasmid was transfer to the pGLO+ tube. The other tube will be the pGLO-, the Escherichia coli without the plasmid. The two tubes were moved into an ice bucket and left there for 10 minutes. Then the tubes were put into a 42?

C water bath for 50 seconds and after back to the ice bucket for 2 minutes more. After the two minutes had passed, a 300 microliters aliquot of LB broth was added to the two test tubes. By adding the LB broth, the CaCl2 solution was also inserted in the tubes with the E. coli. Right after it the tubes were shook for ten minutes in a 37? C shaker. There were gather 4 petri plates, one with LB media, two with LB amp(ampicillin), and the last one with LB amp ara(arabinose sugar). After the 10 minutes each plate was given an aliquot of 100 microliters with one of the E. coli of the eppie tubes.

The LB plate and LB amp had the pGLO- and the other two plates, LB amp ara and LB amp, had the pGLO+. After this step it’s done the plates are prepared to be incubated at 37? C for two days and reveal the results of the induced transformation. LAB 9: TRANSFORMATION PROCEDURE Results The results for this experiment were a bit ambiguous but still recognizable and pretty clear. All of these plates were seen under UV light. In the LB plate pGLO- , after the incubation, there was found a lawn of Escherichia coli colonies that looked green because of the light. The LB amp plate with the pGLO- bacteria, the E. oli did not seem like it grow on it, the media just looked green. A count of 172 colonies that looked green, was found in the LB amp pGLO+ plate, this plate had ampicillin. In the LB ara amp media plate there were found 251 colonies of E. coli. In this plate the colonies looked fluorescent green under the UV light, the only plate. In a scale of growth from larger to smaller, the first in line would be the LB, then LB ara amp, proceeds LB amp (pGLO+), and last one LB amp (pGLO-). Table 1. 1 Results oftransformationof E. coli withpGLO plasmid mediapGLO+pGLO-color(under UV light)growth

LB -Yesgreenlawn of colonies LB amp-yesmedia look greenno growth LB ampyes-green172 colonies LB amp arayes-fluorescent green251 colonies -= n/a Discussion The results obtained in this experiment were as expected. The gfp should had been expressed under the presence of arabinose sugar and then under the UV light would fluoresce. The bla gene was expected to be expressed in the presence of ampicillin molecules. The LB pGLO- plate was a control plate meaning that this plate set a reference parameter to compare the results after the transformation. In this plate the growth of the E. oli was in a vast amount since this is a general media target for growth. In the LB amp pGLO- plate, the other control, the E. coli was not transformed with the plasmid, so in presence of the ampicillin the natural behavior of the bacteria is that is susceptible to it. In another hand, the plate of LB amp pGLO+ presented growth meaning that the bacteria took up the plasmid and was able to expressed the genes by an induce transformation. The result being that the transformed E. coli is now resistant to the ampicillin. The last plate, LB amp ara pGLO+, appeared with 251 fluorescent green colonies under the UV light.

The reason for it is that the bacteria took up the pGLO plasmid and when the E. coli was in the presence of arabinose and ampicillin, the bacteria could fluoresce green and be resistant to ampicillin which naturally the E. coli does not possess this genes. When this last plate is compared with the control plates it can be confirmed that the procedure done in this experiment was effective as hoped. The arabinose sugar is the intriguer that turns on the genes under the ara promoter. So when the gfp under this promoter turns on, all the other genes under the same promoter will expressed in the cell also.

No real noticeable source of error was found during the experiment since the results obtained were completely expected based in the information of the procedure. New studies are being made constantly and this transformation technique is widely used in the field of biotechnology. In the study of Plasmid DNA Transformation in Escherichia Coli: Effect of Heat Shock Temperature, Duration, and Cold Incubation of CaCl2 Treated Cells, the experiment was based on how much quantitative is the difference between different variables possible to reach for the best optimum environment to exploit to the maximum the use of this technique.

These results suggest that a heat shock pulse of 30 sec at 42°C followed by a 10 min ice incubation step are ideal parameters to obtain maximum transformation efficiency, also suggest that post heat shock cold incubation step is also an important factor and enhances transformation of E. coli significantly (Singh 561) The relevance of this paper on the experiment performed and discussed previously is big. The results of Singh’s experiment helps our experiment in enhancing the correctness of our results and lowering the possible errors that can surge.

Also it can be a great reference of how to determine the optimum conditions of a specific bacteria which would contribute in other research fields. Citations Anh-Hue T. Tu. Transformation of Escherichia coli Made Competent by Calcium Chloride Protocol. Microbe Library. American Society of Microbiology. October 25, 2012. Web. November 10, 2012 Garcia-Cayuela, Tomas,. Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli. 172. Applied Genetics and Molecular Biotechnology.

Pdf Panja, Subrata,. Aich, Pulakesh,. Jana, Bimal,. Basu, Tarakdas. How does plasmid DNA penetrate cell membranes in arti? cial transformation process of Escherichia coli? 25(5): 411 Molecular Membrane Biology, August 2008. Pdf. Singh, Mahipal,. Yadav, Arpita. Ma, Xiaoling. Amoah, Eugene. Plasmid DNA Transformation in Escherichia Coli: Effect of Heat Shock Temperature, Duration, and Cold Incubation of CaCl2 Treated Cells. Volume 6 Number 4, 2010. 561– 562 International Journal of Biotechnology and Biochemistry. Pdf.

Categories
Free Essays

Biochemical Action of Bacteria

OBJECTIVE: 1. To distinguish the bacteria abilities to metabolize various substrates and end products formed. 2. To observe the growth of different bacteria species in term of structures and its morphology based on different chemical substance applied. 3. To observe physiological and immunological properties utilized by different species of bacteria. INTRODUCTION: Bacteria biochemical testing can determine the types and numbers in terms of colony forming units of bacteria present in a sample of different chemical. The testing could be focused on a specific type of bacteria, medical bacteria or a broad range of environmental bacteria.

Since bacteria are present in virtually any environment, it’s important to be clear why the testing is being performed. The more specific the testing is the better and the easier it is to interpret the results. Numbers and types of bacteria that should be a cause for concern depends upon several factors, including the type of bacteria present and the type of samples. Escherichia coli are one of the main species of bacteria living in the lower intestines of mammals. E. coli can be found in the intestinal tract of warm-blooded animals. The presence of E. coli in foods is considered to be an indication of fecal contamination.

Staphylococcus organisms are commonly found in the environment. Several species of Staphylococcus are found on the skin, intestines, nasal passages, etc. of warm-blooded animals. Some species of Staphylococcus, particularly Staphylococcus aureus can be pathogenic are capable of causing illness. Pseudomonas aeruginosa is widely distributed in soil, water and plants. It survives in hot tubs, whirlpools, contact lens solution, sinks and showers. It can cause a number of opportunistic infections including infections of the skin, external ear canal and of the eye.

Nitrifying bacteria recycle organic nitrogenous materials from ammonium (the endpoint for the decomposition of proteins) to nitrates. Their presence can indicate that the water may have been polluted by nitrogen-rich organics from sources such as compromised septic tanks, sewage systems, industrial and hazardous waste sites and is undergoing an aerobic form of degradation. The presence of denitrifying bacteria can indicate that the water has been polluted by nitrogen-rich organics from sources such as compromised septic tanks, sewage systems, industrial and hazardous waste sites. MATERIALS: 1. Nutrient broth cultures of Escherichia coli . Nutrient broth cultures of Serratia marcescens 3. Nutrient broth cultures of Salmonella typhimurium 4. Nutrient broth cultures of Bacillus subtilis 5. Nutrient broth cultures of Klebsiella spp. 6. Nutrient broth cultures of Streptococcus spp. 7. Nutrient broth cultures of Staphylococcus aurieus 8. Nutrient broth cultures of Proteus vulgaris 9. Nutrient broth cultures of Pseudomonas fluorescens 10. Parafilm tape 11. Inoculating loops 12. Gloves 13. Incubator 14. Nutrient agar plate 15. Nutrient agar slants 16. Starch agar plates 17. Gelatine agar plates 18. 2 tubes Clark’s-Lub medium (MR-VP medium) 19. Tryptone broth 20. 3 Kigler’ slant 21. 5 tubes nitrate broth ( 0. 1% KNO3) 22. 5 urea broth 23. Tube containing 10ml of sterile saline 24. Glucose broths with Durham tubes and phenol red indicator 25. Lactose broths with Durham tubes and phenol red indicator 26. Sucrose broths with Durham tubes and phenol red indicator 27. Gram’s iodine 28. Kovac’s indol reagent 29. Mercuric chloride solution 30. KOH-creatine solution or 40% KOH 31. F&R reagent 32. Nessler’s reagent PROCEDURE: A. CARBOHYDRATE METABOLISM 1. Fermentation of sugars Materials: 1. Glucose broths with Durham tubes and phenol red indicator 2.

Lactose broths with Durham tubes and phenol red indicator 3. Sucrose broths with Durham tubes and phenol red indicator 4. 18 hour nutrient broth cultures of E. coli and S. typhimurium Procedure: 1) The small bottles of different sugars were inoculated with a loopfuls of E. coli and Salmonella spp. 2) The tubes were labelled and incubate at 37oC for 24 hours 3) All observations were recorded for presence of acid or gas production. 2. Hydrolysis of starch Materials: 1. Starch agar plates 2. Broth agar cultures of B. subtilis and E. coli Procedure: 1) Starch plate was streaked with E. coli in for sections and repeated for B. ubtilis bacteria in other starch plate. 2) The plates were secured with parafilm, labelled and inoculated at 37oC for 24 hours. The following day 1) The plates were tested for starch hydrolysis by flooding the pates with Gram’s iodine. 2) The plates were examined and the colonies that showed clear uncoloured zones in contrast with the blue-black background of the starch-iodine complex were noted. 3) The extent of the zones of hydrolysis indicated either the reddish colour zones were seen. 4) All results and observations were recorded. B. PROTEIN AND AMINO ACID METABOLIM 1. Indole test Materials: 1. Broth cultures of B. ubtilis, E. coli, and S. typhimurium 2. 3 tubes of tryptone broth 3. Kovac’s indole test reagent Procedures: 1) The peptone water was inoculated with a loopfuls of the test organism. 2) The tube was labelled and incubated for 24 hours. The following day 1) The tubes were added with a few drops of Kovac’s indole reagent (dimethylaminobenzaldehyde) 2) The red or dark color indicates the presence of indole. 4. Hydrogen sulphide Materials: 1. Broth cultures of B. subtilis, E. coli, and S. typhimurium 2. 3 Kigler’s slant Procedures: 1) The Kigler’s slant was inoculated with a loopfuls of the test organism by the stab method. ) The tube was labelled and incubated for 24 hours. The following day 3) The Kigler’ slant was observed for production of H2S where the black precipitate along the line of growth in the Kigler’s slants indicated the H2S have been produced. 4) The observations were recorded. 3. Gelatine hydrolysis test Materials: 1. Broth cultures of B. subtilis, E. coli, and S. typhimurium 2. Gelatine agar plates 3. Mercuric chloride solution Procedures: 3) The gelatine agar plates were inoculated with a loopfuls of the test organism with a single streak at the centre of the plates. ) The plates were secured with parafilm, labelled and incubated for 24 hours. The following day 5) The plates were flooded with mercuric chloride solution. 6) The medium become opaque in regions that still contain gelatine and clear regions where gelatine has been hydrolysed. C. VOGES-PROSKAUER TEST Materials: 1. Broth cultures of E. coli, and Klebsiella spp. 2. 2 tubes of Clark-Lub’s medium (MR-VP medium) 3. KOH-creatine solution Procedures: 1) The tubes of Clark-Lub’s medium (MR-VP medium) were inoculated with a loopfuls of the test organism. 2) The tubes were labelled and incubated for 24 hours.

The following day 1) The tubes were tested with Voges-Proskauer test. 2) The 0. 5ml of KOH-creatine solutuin was addd. 3) The tube was shaked vigorously for 30 seconds. 4) The red or pink color indicates the presence of acetoin. D. CATALASE TEST Materials: 1. Broth cultures of Streptococcus spp. and Staphylococcus aureus. 2. Nutrient agar slant Procedures: 1) The nutrient agar slant was inoculated with a loopfuls of the test organism. 2) The tube was labelled and incubated for 24 hours. The following day 1) The tubes were tested with catalase test by adding several drops of a 5% solution of hydrogen peroxide. ) The vigorous bubbling indicates the presence of oxygen. E. NITRATE REDUCTION TEST Materials: 1. Broth cultures of E. coli, Proteus vugaris, Serratia marcescens, Pseudomonas fluorescens. 2. 5 tubes containing nitrate broth (0. 1% KNO3) 3. Nitrate test reagent Procedures: 1) The nitrate broth was inoculated with a loopfuls of the test organism. 2) The tube was labelled and incubated for 24 hours. The following day 1) The tubes were tested with 1ml of Follet and Ratcliff’s (F&R reagent) 2) The orange or brown color indicates the presence of nitrate. 3) The absent of nitrate indicates that: a.

There has been no nitrate reduction b. The reduction has proceeded beyond that nitrate stage. 4) The absent of orange or brown color were further tested with small amount of cadmium to the tube. If nitrate still present, it will be catalytically change to nitrate which will then reacts with the F&R reagent in the tube. 5) In the absent of a positive nitrate result, the bubbles f H2 gas was observed in the Durhams tube OR 6) The samples were tested with 1ml of Nessler’s reagent. The brown or orange color indicates the presence of ammonia. F. UREASE TEST Materials: 1. Broth cultures of E. coli, P. vugaris, S. arcescens, P. fluorescens. 2. 5 urea broth with indicator Procedures: 1) The urea broth was inoculated with a loopfuls of the test organism. 2) The tube was labelled and incubated for 24 hours. The following day 1) The urease-positive organism produced in intense red/purple coloration of the medium after incubation. 2) All observations were recorded. RESULTS AND OBSERVATION: Test| Observation(After 24 hours incubation)| Description| A. Carbohydrate Test 1. Fermentation of starchDurham tubes and phenol-red indicator. 2. Hydrolysis of starch| Glucose: Lactose: Sucrose: Starch agar plates:B. ubtilisE. coli| * Positive result for E. coli as tube turn yellow * Positive result for S. typhimium as tube turn yellow * Positive result for E. coli as tube turn yellow * No gas produced by S. typhimium because the tube turns red. * No gas produced by E. coli because the tube is slightly red. * Positive result for S. typhimium as tube turn yellow * Positive zone of clearing. * Negative zone of clearing. | B. Protein And Amino Acid Metabolism 1. Indole test 2. Hydrogen disulphide 3. Gelatine hydrolysis test| Tryptone broth:B. subtilisE. coli. S. typhimuriumKigler’s slant:B. subtilisE. oli. S. typhimuriumGelatine agar plates:B. subtilisE. coli. S. typhimurium| * Negative Indole tests no color change. * Bright fuschia at the interface is positive test for Indole. * Negative Indole tests no color change. * Black precipitate form shows positive sulphur reduction. * Negative reaction. * Positive reaction forming the black precipitate. * Positive hydrolysis of gelatine into amino acid to be used as nutrients/gelatinase. * Negative hydrolysis of gelatine. * Negative hydrolysis of gelatine| C. Voges- Proskaeur’s Test| MR-VP medium:E. coli. Klebsiella spp. | * Negative results of E. oli * Positive results Klebsiella spp. | D. Catalase Test| Nutrient agar slant:S. aureusStreptococcus spp. | S. aureus * Positive catalase reaction because present of bubblesStreptococcus spp. * Negative catalase reaction no bubbles present. | E. Nitrate Reduction Test| Nitrate broth:E. coliP. vulgarisS. marcescensP. fluorenscens| * No color change after denitrification of ammonia. * No color change after denitrification of ammonia. * Turns red. Positive nitrate test shows nitrate reductase present. * Turns red but negative catalase test. | F. Urease Test| Urea broth:E. coliP. vulgarisS. marcescensP. luorenscens| * Negative urease test because the tube remain purple. * P. vulgaris show positive urease test from yellow to pinkish. * S. marcescens show negative urease test because the color remain purple. * P. fluorenscens show negative urease test because the color remain purple. | DISCUSSION: Biochemical tests of bacteria oobjectively to test the metabolism of carbohydrate and related products of different bacteria species, test specific breakdown of products through color changes and gas produced. Besides that, the ability of bacteria utilizes a specific substance and the metabolism of protein and amino acid by bacteria.

A. CARBOHYDRATE TEST Carbohydrate is an organic compound that consists of only carbon, hydrogen and oxygen which is basically the major carbon source of most organisms. Specific carbohydrate can be fermented by organism that incorporated in a medium producing red or acid with gas. Pinkish red color shows positive results where acidic content formed in the tube because carbon dioxide realised if fermentation occur. Negative catabolism of carbohydrate shows by yellow to colourless of Durham’s tube as the solution remain alkaline in the absent of carbon dioxide gas.

Gas production can be seen as bubbles in Durham’s tube. Central carbohydrate metabolism or the breakdown of sugars into smaller compounds accompanied by the production of ATP and reduction of coenzymes, follows one of several pathway. Carbohydrate utilization and fermentation will be assessed by growing cells without shaking (aeration) in defined media containing a single carbohydrate. Acid products of sugar fermentation will cause a noticeable color change in the pH indicator included in the medium.

Sugar fermentation does not produce alkaline product, however non-fermentative hydrolysis of amino acids in the peptone, present in most fermentation media, may give an alkaline reaction, which will also cause a color change in the pH indicator. Gas production, H2 in particular, can be determined by placing a small, inverted Durham tube in the test medium. If gas is produced, it is trapped in the Durham tube and can be seen as a bubble. Hydrogen sulfide (H2S) is produced by bacterial anaerobic degradation of the two sulfur-containing amino acids, cysteine and methionine.

Hydrogen sulfide is released as a by-product when carbon and nitrogen atoms in the amino acids are consumed as nutrients by the cells. Under anaerobic conditions the sulfhydryl (-SH) group on cysteine is reduced by cysteine desulfurase. Ferrous ammonium sulfate-indicator. H2S reacts with ferrous sulfate forming the black precipitate Sodium thiosulfate is reduced to sulphite/thiosulfate The Kligler’s Iron test is used to detect liberation of H2S gas by bacteria growing on an excess of these sulfur-containing amino acids. The agar contains high levels of peptones or sources of cysteine and methionine and ferrous sulfate as an indicator.

When H2S is produced, the ferrous ion reacts with it to give ferrous sulfide, an insoluble black precipitate. In starch hydrolysis test Iodine must be on the plate to visualize the zone of clearing surrounding the bacteria. This zone indicates starch was broken down to dextrins, maltose, and glucose. B. PROTEIN AND AMINO ACID METABOLIM Indole test measures the ability of bacteria to split indole from tryptophan molecule but in term of biochemistry, Indole test is one of the metabolic degradation products of the amino acid tryophan.

Bacteria that possess the enzyme trytophanase are capable of hydrolysing and deaminating tryptophan with the production of Indole, pyruvic acid and ammonia. Positive reaction showed by E. coli, P. vulgaris and negative results observed in Klebsiella and Salmonella from observation in the Indole test. Development of fuchsia red color at the interface of the reagent and the broth within seconds after adding the reagent is indicative of the presence of Indole and is a positive test. Kovac’s reagent detects if tryptophan has been hydrolyzed to indol or tryptophanase.

Gelatin is the protein derived from the animal protein collagen, has been used as a solidifying agent in food for a long time besides nutrient gelatine as an early type of solid growth medium. One problem is that many bacteria have the ability to hydrolyze or liquefy the gelatin. This gelatin liquefaction ability forms the basis for this test. C. VOGES-PROSKAUER TEST The production of acetoin by bacteria is perform through Voges Proskauer Test to determine the ability of the organisms to produce neutral end product acetyl methyl carbinol (acetoin) from glucose fermentation.

Negative results gained from E. coli meanwhile positive reaction gives by. Changing of color to red pinkish color at the surface of the medium indicated positive results and yellow color at the surface of the medium show negative reaction. The KOH reagent should not be excessively added to the sample because excess KOH may mask weak VP positive reactions. The MR test will be positive for organisms that have complete pathways for mixed acid fermentation. The Voges-Proskauer (VP) test determines whether a specific neutral metabolic intermediate, acetoin, has been produced instead of acid from glucose.

Acetoin is the last intermediate in the butanediol pathway, which is a common fermentation pathway in B. subtilis. The tests are complementary in the sense that often a bacterium will give a positive reaction for one test and a negative reaction for the other. The three possible patterns of results where the acetoin fermentation pathway, detected by the VP test, two molecules of pyruvate condense and two molecules of CO2 are released. The 4 carbon intermediate that is formed, acetoin, contains a carbonyl group. The acetoin acts as a terminal electron acceptor with the carbonyl group being reduced to a hydroxyl group.

The reduced product, butanediol, is excreted by the bacteria and acetoin is oxidized to diacetyl by alkaline -naphthol, which forms a red complex with creatinine. D. CATALASE TEST Catalase is present in most cytochrome containing aerobic and facultative anaerobic bacteria except Streptococcus spp. Hydrogen peroxide forms as one of the oxidative end product of aerobic carbohydrate metabolism. If hydrogen peroxide allowed accumulating in the bacterial cells it becomes lethal to the bacteria. Catalases help in converting H2O2 to water and oxygen.

In the catalase test performed, Streptococcus spp gives negative reaction as for S. aureus, the positive reaction occurred. One of the by-products of oxidation-reduction in the presence of O2 during aerobic respiration is hydrogen peroxide (H2O2). This compound is highly reactive and must be degraded in the cytoplasm of the cell producing it. It can be especially damaging to molecules of DNA. Most aerobes synthesize the enzyme catalase, which breaks down H2O2 into water and oxygen. The O2 gas is identified by the production of bubbles from a concentrated cell suspension.

The test for catalase is simple and usually very reliable. It is a major method of distinguishing between Staphylococcus (catalase positive), Streptococcus (catalase negative), and Enterococcus (catalase negative), although some strains of Enterococcus faecalis may be positive. Catalase production is generally associated with aerobic organisms, since H2O2 is a toxic by-product of aerobic growth, but not always. E. NITRATE REDUCTION TEST Nitrate reduction test basically test the ability of organism to reduce the nitrate to nitrites of free nitrogen gas.

In order to determine either the bacteria can reduce nitrate, the test organism is inoculated into nitrate reduction broth, undefined medium that contains large amounts of nitrate (KNO3). After incubation, reagent added simultaneously reacts with nitrite and turn to red color, indicating a positive nitrate reduction. If there is no color change at this step, nitrite is absent. If the nitrate is unreduced and till in its original form, this would be a negative nitrate reduction result. However it is possible that the nitrate was reduced to nitrite but has been further reduced to ammonia or nitrogen gas.

This would be recorded as positive nitrate reduction result. Under anaerobic conditions, some bacteria are able to use nitrate (NO3-) as an external terminal electron acceptor. This kind of metabolism is analogous to the use of oxygen as a terminal electron acceptor by aerobic organisms and is called anaerobic respiration. Nitrate is an oxidized compound and there are several steps possible in its reduction. The initial step is the reduction of nitrate (NO3-) to nitrite (NO2-). Several possible products can be made from further reduction of nitrite. Possible reduced end products include the following N2, NH3 (ammonia), N2O (nitrous oxide).

Bacteria vary in their ability to perform these reactions, a useful characteristic for identification. A medium that will support growth must be used and the cells must be grown anaerobically. Growth in the presence of oxygen will decrease or eliminate nitrate reduction. There are many possible end products of nitrate reduction such as nitrite, nitrogen gas (N2), nitrous oxides, ammonia, and hydroxylamine. The disappearance of nitrate or the appearance of the end products. The test relies on the production of nitrous acid from the nitrite. This, in turn, reacts with the iodide in the reagent to produce iodine.

The iodine then reacts with the starch in the reagent to produce a blue color. Since some of the possible products of NO3- reduction are gaseous, a Durham tube is sometimes inverted in the culture tube to trap gases. This being the case, it is important to pre-test the medium to ensure no detectable nitrite is present at the beginning, and, in the case of a negative test, to reduce any nitrate to nitrite to determine whether the nitrite was also reduced. If nitrite is produced, it reacts with hemoglobin to give a bright red color, instead of the dark red color of hemoglobin.

It is this reaction that is responsible for the color of meats, such as hot dogs, which are preserved with sodium nitrite. The blood agar test has the advantage of no color change occurring if the nitrite is further reduced. F. UREASE TEST Urease test mainly highlighted to determine the ability of the organism to split urea forming 2 molecules of ammonia by the action of the enzyme Urease with resulting alkalinity. Negative reaction shown by E. coli meanwhile Klebsiella spp. shows positive result. Extra precaution needed because both the urease test medium depend upon the demonstration of alkalinity that not specific for urease.

Moreover the protein hydrolysis may result I alkalinity hence false positive may be seen in Pseudomonas. The false positivity can be eliminated by control test using the same medium without urea as recommendation. Urea is a nitrogenous waste product of animals. Some bacteria can cleaved it to produce carbon dioxide and ammonia. The ammonia is a nitrogen source for amino acid biosynthesis as well as for synthesis of other nitrogen-containing molecules in the cell. The urease test was devised to distinguish Proteus species from other enterics.

The medium described here is buffered enough so that weak urease producers appear negative. The production of ammonia raises the pH of the medium. The indicator phenol red is present in the broth. Phenol red is orange-yellow at pH below than 6. 8, and turns bright pinkish-red at pH higher than 8. 1. Hence, a positive urea test is denoted by the change of medium color from yellow to pinkish red. CONCLUSION: Based on the laboratory, different bacteria species have different abilities to metabolize various substrates and end products formed were able to be observed and distinguished.

Categories
Free Essays

Lab: the Bacteria Around You

Lab: The Bacteria Around You James Brunet Ms Owen October 14th, 2012 Part 1 Purpose To culture and observe the various types of bacteria found around Canterbury High School. Materials and Methods Refer to pages 422-425 of Biology 11 McGraw-Hill Ryerson and the handout “Gram Staining Procedure”. Observations Table 1: Locations of Bacteria Samples Quadrant| Location of Sample Obtained| 1| Floor| 2| Water fountain head| 3| Auditorium Chair| 4| Inside of Boys’ Bathroom Door Handle|

Table 2: Growth of Bacteria from Various Locations Around CHS after 48h in Incubator Quadrant| Total Number of Colonies| Description of Colonies| Number of Colonies| 1| 1| Irregular, flat, and lobate. Occupies entire quadrant. | 1 | 2| About 8| Milky-white coloured, punctiform, and entire. | 6| | | Milky-white coloured, punctiform, and curled. | 2| 3| 10| Milky, punctiform, and entire. | 8| | | Yellow, punctiform, and entire. | 2| 4| 8| Milky, irregular, lobate, and raised. | 1| | | Milky, punctiform, and entire. | 4| | Milky, punctiform, and curled. | 2| | | Clear, flat, circular, and undulate. | 1| Discussion What areas around the school appeared to have the most bacteria? The least? Suggest reasons for these findings. The area around the school that appeared to have the most bacteria was the inside of the boy’s bathroom door handle. Not only did it have the most diverse range of bacteria (four different types), it also had the 2nd highest number of colonies! The area around the school with the least bacteria was, in my opinion, the floor.

Although the single colony there grew very large, this was probably due to lack of competition, as there were no other colonies present. This seems to indicate that there is actually less diversity of bacteria on the floor than on a door handle. I think that the door handle appears to have more bacteria for two main reasons. Firstly, the door handle is gripped by students exiting the bathroom. Some of these students may not have washed their hands, leading to bacteria being transferred from person to handle constantly. Secondly, these handles are rarely, if ever cleaned, while the floors are cleaned on a daily basis.

Describe the conditions necessary for bacterial growth. Bacteria need food, moisture, warmth, and time to grow. The agar plate provides the food and some moisture, the incubator provides growth, and if it is an expensive unit, moisture as well, and you as the student provides the time. Describe two factors that may limit bacterial growth. A lack of moisture may limit bacterial growth. Instead of multiplying, the bacteria may die. As well, a less than optimal temperature may limit, and perhaps completely stop, bacterial growth.

Temperatures outside of the range of 4°C-60°C (The bacterial “danger zone”) will stop most bacterial reproduction and kill many species of bacteria. However, some bacteria can survive with very little moisture for extended periods of time and thrive outside these temperature ranges. Did this experiment have a control? If not, suggest what control you could set up and why? This experiment did not have a control. If I was to set up a control for this experiment, I would leave one of the quadrants clear of any specimen, and use it as a control quadrant.

If I did that, I could tell if bacteria was already present in the agar if the control quadrant grew colonies. Discuss some aspect relating to your samples or the procedure. I would like to retest the floor sample, because the single colony left me thinking that the data was incomplete. I just don’t think there is only one type of bacteria living on the floor. I think I would like to change the procedure, as a control quadrant is vital to the integrity of the experiment! As well, I am definitely not going to open the boy’s bathroom door and then proceed to touch my eyes immediately after. Conclusion

In conclusion, the bacteria from the floor, water fountain, chair, and door handle flourished because of the warmth of the incubator, the food/moisture present in the agar, and the time we gave it. We identified multiple colonies of bacteria by their colours, sizes, shapes, and thicknesses, and also by staining them with Crystal Violet and Safranin. All of these things combined gave our group insight into the conditions necessary for bacterial growth, how to identify colonies, and where bacteria grow most. Part 2 Purpose To test the effectiveness of various disinfectants and antibiotics on limiting bacterial growth.

Materials and Methods Refer to pages 428-429 of Biology 11 McGraw-Hill Ryerson. Observations Quadrant| Type of antibiotic/disinfectant| Size of zone of inhibition| 1| Soap| Huge zone-extends into quadrant 3. | 2| Organic disinfectant| Midsize zone| 3| Bleach+Comet| None| 4| Hand sanitizer| None| Discussion How was the effectiveness of each antibiotic/disinfectant measured? The effectiveness of each antibiotic/disinfectant was measured by looking at the zone of inhibition, the size of the area immediately surrounding the antibiotic that is colony-free.

Which inhibitor was the most effective? Explain. The soap was by far the most effective inhibitor. Its zone of inhibition extended so far that it even reached into a neighbouring quadrant! This means that the soap was extremely effective at stopping bacterial growth. Rank the inhibitors you used by their effectiveness. Explain your reasoning. I found soap to be the most effective inhibitor, and the organic disinfectant to be the second most effective inhibitor, with respect to their zones of inhibition.

I ranked hand sanitizer and bleach+comet as a tie for last place, because they did literally nothing to stop the growth of bacteria. I ranked these inhibitors in this order because I believe that effectiveness can easily be measured by the size of the zone of inhibition. Why is it important for a physician to know the exact identity of the bacteria involved in an infection? It is important for a physician to know the exact identity of the bacteria involved in an infection because different inhibitors work for different bacteria.

If the physician incorrectly identified the bacteria, his prescribed antibiotics may do nothing against the bacterial infection, and the patient’s sickness would actually worsen. This is easily shown by our zone of inhibition experiment, where of the four chosen antibiotics, two of them did absolutely nothing against the bacteria. Conclusion In conclusion, it is important to know how to both identify bacteria using morphological clues as well as it is important to know how to treat said bacteria. Not all bacteria are the same, and not all antibiotics are on the same footing either.

Categories
Free Essays

Bacterial Growth Requirements

Bacteria Growth Requirements Microbiology Life as we now it has ended. What is left you ask? Well it is said the only thing that could survive an incident that could end our known way of life is a roach and a pack or Twinkies. In truth the great survivor would be microorganisms. Microorganisms can survive where most cannot due to their size, nutritional needs, energy requirements, and are very good at adapting to different environments (Black 2008).

Microorganisms require two things to live a long healthy life, and these are physical and nutritional factors. Physical factors include pH, temperature, oxygen concentration, moisture, hydrostatic pressure, osmotic pressure, and radiation (Black 2008). Nutritional factors include carbon, nitrogen, sulfur, phosphorus, trace elements, and sometimes vitamins (Black 2008). For the purpose of this exercise I will focus on E. coli. Pathogenic Escherichia coli will be discussed since it is a common, but dangerous bacterium.

E. coli in humans is found in the intestines. This bacterium is very durable, meaning that it is well-adapted to its habitat. For example, it can grow with glucose being the only food source. This bacterium can also grow with or without O2. If located in anaerobic habitat it can it will use the fermentation process producing mixed acids and gases (Todar 2012). This bacterium has shown that it can also use anaerobic respiration when NO3 or NO2 is available.

Chemicals, pH, temperature, are a few signals that determines how E. coli will respond (Todar 2012). When it senses a change in the environment it can swim toward or away from anything useful or harmful. Temperature can also affect E. coli. A change in temperature allows E. coli to change pore diameter of its outer membrane to accommodate certain nutrients, or to exclude something harmful. E. coli also rations its nutrient supply by taking in account how much is available in its environment.

This means that it will not take in nutrients unless it has enough to feed more bacteria that will be produced (Todar 2012). As you can see, this amazing microbe has the ability to adapt to its environment and in some case overcome. Imagine the microbes that are out there that has not be identified yet. Reference Black, J. (2008). Microbiology principals and explorations. (7th Edition ed. ). Jefferson City: GGS Book Services. Todar, K. (2012). Todars online textbook of bacteriology. Retrieved from http://www. textbookofbacteriology. net/e. coli. html

Categories
Free Essays

Bacterial Growth Requirements

Bacterial Growth Requirements Evelyn Lyle ITT Technical Institute Angela Ask, MPS January 15, 2012 Every organism must find in its environment all of the substances required for energy generation and cellular biosynthesis. The chemicals and elements of this environment that are utilized for bacterial growth are referred to as nutrients. Many bacteria can be identified in the environment by inspection or using genetic techniques. The nutritional requirements of a bacterium such as E Coli are revealed by the cell’s elemental composition.

These elements are found in the form of water, inorganic ions, small molecules and macromolecules which serve either a structural or functional role in the cells. Bacteria thrive by four things oxygen, food (nutrients), warmth and time but two others can be moisture and acidity. Nutrients are needed for energy, nitrogen (for DNA and proteins), phosphorus (for energy), and others. Warmth is needed so the bacteria can stay warm. Oxygen is needed so the bacteria can make energy and time is needed for the bacteria to complete binary fission over and over again. Acidity is needed so the bacteria can survive in its environment.

Highly base or acidic environments may harm the bacteria and hinder its lifespan. In order to survive and grow, microorganisms require a source of energy and nourishment. Bacteria are the most primitive forms of microorganisms but are composed of a great variety of simple and complex molecules and are able to carry out a wide range of chemical transformations. Depending on their requirements and the source of energy used they are classified into different nutritional groups. Most microorganisms grow well at the normal temperatures favored by man, higher plants and animals.

Certain bacteria grow at temperatures (extreme heat or cold) at which few higher organisms can survive. Most bacteria grow best in an environment with a narrow pH range near neutrality between pH 6. 5 and 7. 5. Microbes contain approximately 80-90% water and I f placed in a solution with a higher solute concentration will lose water which causes shrinkage of the cell. Some bacteria have adapted so well to high salt concentrations that they actually require them for growth. Nitrogen and phosphorus are particularly critical because they often control the rates of photosynthesis.

Carbon is significantly more abundant than either of them and oxygen and sulfur are more abundant that phosphorous. Nitrogen and phosphorous are less available to plants relative to their growth requirements than are other elements. Phosphorus is often in short supply and limits plant and algae growth. Nitrogen is a major constituent of all proteins and of all living organisms. A lack of nitrogen can limit growth of plants, since nearly three quarters of its atmosphere consists of natural gas, N2. REFERENCES A New Way to Look at Microorganisms. (n. d). American Scientist, 93(6), 514.

Categories
Free Essays

Microbiology: Bacteria and Fresh Yogurt Slide

Bacterial Morphology Demonica Britt Microbiology DL1 March 23, 2013 Abstract This lab was performed to identify and familiarize with a microscope while precisely observing various bacterial shapes and their arrangements in different types of specimens of bacteria. The microscope parts and capabilities were clearly identified and used successfully and the bacteria were clearly illustrated showing the bacterial shapes and arrangements with all the appropriate magnification being utilized.

Through various magnifications using 10x, 40x and 100x oil immersion lenses, the bacteria specimens, along with fresh and prepared yogurt, demonstrated full visual optical views of their shapes and how the different types were displayed at different levels of magnification. Purpose The purpose of the experiment was to gain full knowledge and experience of operating a microscope while being able to successfully visualize different types of bacterial and yogurt specimen’s shapes and arrangements using several magnification techniques by way of 10x, 40x,100x oil immersion lenses and a light source.

The main purpose was to observe the shapes and arrangements of microbial bacteria and yogurt. Procedure The lab involved self-provided and labpaq materials to perform several exercises to obtain the purpose of the lab. The lab began with the proper identification of all components of the microscope and their functions. This allowed for preparation of the objective of being able to view specimens at various magnification levels and recognizing their different shapes and how they are arranged contingent upon those identified within the lab itself and the microbiology textbook.

Several different slides were observed under 10x and 40x lens magnification: Paramecium conjugation, Yeast, Amoeba Proteus, Ascaris eggs, Anabaena, and Penicillium. This allowed vivid illustrations of the specimens notating their shapes and how they are arranged. The bacteria were observed through the eyepiece at the appropriate focus, resolution, and contrast for maximum visibility. The next part of the lab exercise was observance under an 100x oil immersion lens for more prepared slides: Bacteria Coccus form, Bacteria spirillum, and Bacteria Bacillus form while still maintaining to observe the shapes and arrangements.

Additionally, the fresh yogurt slide that was sitting for 24 hours in a dark, warm location was obtained for the next part of the lab experiment. The fresh yogurt slide was prepared by using a toothpick to place a small amount onto a fresh, clean slide with a slide cover placed on top. This was observed for comparison to the prepared yogurt slide included in the lab for any variations in forms. Upon completion of performing the lab, the prepared slides were safely put away, fresh slide washed carefully, fresh yogurt specimen safely discarded, and the microscope cleaned and returned to be stored with the protective cover.

Data/Observations – (Data Tables & Photos of Labeled Pics & Observations) The bacteria slides clearly displayed the various types of bacteria shapes and showed how each follow a specified arrangement. Under the lowest magnification the object is relatively smaller and not as easy to see the full format. Whereas the higher the magnification, the bigger and more enhanced the view of the bacteria becomes making the shapes and arrangements relatively obvious. It appeared to become clearer the bigger the object projected to my eye.

It became life size in a sense where as it was an image that could be clearly defined, described and duplicated if necessary. The fresh yogurt slide that was set for 24 hours was a more enhanced feature for observing bacteria in yogurt. Its view was very detailed and its shape more recognizable. While the prepared yogurt slide was a more faint view and the color appearing duller. It was visible to me that bacteria in yogurt was more spherical in shape, cocci. Results A. What are the advantages of using bleach as a disinfectant? The disadvantages? The advantages of using 70% alcohol?

The disadvantages? Bleach is a common household disinfectant that kills 99. 9 percent of germs whereas others cannot approach this effectiveness. It can be used to sanitize. It can be a disadvantage as it can be inactivated by presence of an organic matter and it has a strong odor and it has a short life in the liquid form that can be sensitive to heat and sunlight. The advantages of using 70% bleach is that it can be capable of killing most bacteria which is safe for skin contact and it prevents dehydration and the alcohol part of it affect the cells in various ways.

Some disadvantages are that they are hazardous which contain compounds that are not safe and toxic to human form. B. List three reasons why you might choose to stain a particular slide rather than view it as a wet mount. C. Define the following terms: Chromophore: Acidic Dye: Basic Dye: D. What is the difference between direct and indirect staining? E. What is heat fixing? F. Why is it necessary to ensure that your specimens are completely air dried prior to heat fixing? G.

Describe what you observed in your plaque smear wet mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences? H. Describe what you observed in your cheek smear wet mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences? I. Describe what you observed in your yeast wet mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences? J. Were the cell types the same in all three specimen sets:  yeast, laque, and cheek? How were they similar? How were they different? Conclusion/Discussion Upon performing and completing the experiment I learned that the microscope is a very delicate tool that allows the capability of viewing specimens too small for the human eye. With adjusting the focus, contrast, and resolution, the bacteria become more visible to the eye. On top of that, viewing the specifications at different magnifications the bacteria shapes and arrangements become more present within the specimen.

Bacteria comes in different forms and shapes and just by arrangement alone, they can be classified morphologically. It was also visual that there are differences in a fresh slide containing bacteria compared with a slide already prepared. I did not expect to see the differences so vividly displayed, but after using the microscope it was determined that anything not visible to the naked eye still has the capability to be seen and the microscope is the perfect tool to use to be able to do so.

Categories
Free Essays

Fighting Bacterial Growth

Fighting Bacterial Growth The purpose of this lab was to determine the effectiveness of antiseptics, disinfectants, and antibiotics on bacteria. The hypothesis was that if bleach was used, it would be the most effective because bleach is commonly used to clean and disinfect various things. The variables that were tested were antibacterial soap and Scope mouthwash for the antiseptics; bleach and ammonia for the disinfectant; and Cipro, erythromycin, and tetracycline for the antibiotics.

All of these chemicals were used on the bacteria M. luteus. Two Petri dishes were covered in the bacteria and split into four quadrants, in which each had a disc containing one of the chemicals stated above. One quadrant was left alone with no chemicals for the control group. The dishes were then left for the bacteria to grow, and once obtained again it was obvious that some of the bacteria was killed by the chemicals.

In individual data, there was a zone of inhibition of 3mm in the antibacterial soap; 10mm in the Scope mouthwash; 2mm in the bleach; no zone of inhibition around the ammonia; 10mm in both the erythromycin and the tetracycline, and 15mm for the Cipro. The average length of the halo of inhibition in antiseptics was 8 mm in the E. coli, and 6 mm in the M. luteus. The average length of the halo of inhibition in the disinfectants was 12mm and 11mm respectively. For the antibiotics, it was 7mm and 9mm respectively.

The data represented the hypothesis because for both the E. coli and the M. luteus, the largest zone of inhibition was in the disinfectant; and more specifically, the averages were 28mm in the E. coli and 18mm in the M. luteus in the bleach. In some Petri dishes, the bleach also killed bacteria in the other quadrants, indicating that it killed a lot of bacteria. This also affected some measurements for the other chemicals, because the zone of inhibition for the other chemicals around the bleach could have been caused by the bleach instead of the other chemical.

Another error was that since these Petri dishes were left out for 2 days, there was re-growth in the bacteria in and around the zones of inhibition, like the ammonia in the individual data. In the lab, the chemicals were tested on bacteria to see how much of the bacteria will get killed. What kind of items then would create the most bacterial growth? If bleach was used on different kinds of bacteria, which kinds of bacteria would be most affected by the bleach, and which bacteria will be the least affected by the bleach?