THE UNIVERSITY OF ZAMBIA

SCHOOL OF VETERINARY MEDICINE

PARACLINICAL STUDIES DEPARTMENT

NAME:

BERTHA CHITAMBO

COMPUTER...
IDENTIFICATION OF ENTEROBACTRIA BY BIOCHEMICAL TESTS
AIM
To identify enterobacteriaceae in culture A and B by biochemical ...
Agar contains three fermentative sugars, lactose and sucrose in 1% concentration and glucose in a
concentration of 0.1%.
C...
MATERIALS
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Cultures A & B
Bunsen burner
Inoculating wire –straight wire
Inoculating loop
Urea agar...
The straight wire was first sterilized by heating till red hot over a Bunsen burner flame. The mouth
part of the test tube...
DISCUSSION

The four biochemical tests were carried out in order to differentiate and identify enterobacteria
and in this ...
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Bacteriology&immunology lab3

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Bacteriology&immunology lab3

  1. 1. THE UNIVERSITY OF ZAMBIA SCHOOL OF VETERINARY MEDICINE PARACLINICAL STUDIES DEPARTMENT NAME: BERTHA CHITAMBO COMPUTER NUMBER: 10032967 COURSE CODE: VMP 4300 MICROBIOLOGY/IMMUNOLOGY LAB NUMBER 3: IDENTIFICATION OF ENTEROBACTERIA BY BIOCHEMICAL TESTS ATTENTION: MR. MUBITA DUE DATE: 3/12/2013
  2. 2. IDENTIFICATION OF ENTEROBACTRIA BY BIOCHEMICAL TESTS AIM To identify enterobacteriaceae in culture A and B by biochemical tests INTRODUCTION The identification of bacteria is a careful and systemic process that uses many different techniques to narrow down the types of bacteria that are present in an unknown bacterial culture. It produces benefits for many aspects of the research of microorganisms and helps the physician to correctly treat patients. Various steps are involved in the identification of unknown bacteria in the laboratory. Isolation is the first one. The importance of this step is to isolate pure colonies of bacteria. The streak plate is a quantitative isolation method. The inoculation of the culture is made on the agar surface by back and forth streaking with the inoculation loop over the solid agar surface. This will make a dilution gradient across the agar plate. The characteristic features in the identification of unknown bacteria being; the shape, size, elevation, surface, edges, color, degree of growth and nature. The second method being microscopic and staining reactions method, Staining is a simple basic technique that is used to identify microorganism. For example, Gram staining is a differentialstaining technique that imparts different colors to different bacteria or bacterial structures. Usually it differentiates bacteria into two groups: Gram positive and Gram negative bacteria. The primary stain crystal violet and mordent iodine form strong Crystal violet iodine (CVI) complex. Gram positive bacterial cells due to their thick peptidoglycan layer will retain the CVI complex even after it is subjected to decolorization by acetone or alcohol. Hence, the counter stain safranin has no action on the gram positive cells. But in the case of gram negative bacterial cells, the thin peptidoglycan layer and more lipid contents in the cell will easily make them susceptible to the action of the decolorizer and hence the CVI complex is easily washed out and therefore the gram negative bacteria cells will retain the color of the counter stain safranin. Hence, after gram staining, the gram positive cells appear purple and the gram negative cells will appear pink. Then it can be said that the study of morphological features and staining characteristics help in the preliminary identification of the isolate. Biochemical reactions are also used in the identification of enteric bacteria. Gram negative enteric bacilli play an important role in the contamination of food. Hence, they are the main causative agents of intestinal infection. Gram negative family includes Shigella, Salmonella, Proteus, Klebsiella, Esherichia, and Enterobacter. Four biochemical tests were carried out in this practical, these being: Triple sugar – iron (TSI) test, Citrate test, SIM (H2S, Indole, Motility) and the Urease test. TSI test is designed to differentiate among the different groups or genera of the Enterobacteriaceae, which are all gram negative bacilli capable of fermenting glucose with the production of acid, and to distinguish them from other gram negative intestinal bacilli. This differentiation is based on the differences in carbohydrate fermentation patterns and hydrogen sulfide production by the various groups of intestinal organisms. Carbohydrate fermentation is detected by the presence of a gas and a visible color change (from red to yellow) of the pH indicator, phenol red. The production of hydrogen sulfide is indicted by the presence of a precipitate that blackens the medium in the butt of the tube. TSI
  3. 3. Agar contains three fermentative sugars, lactose and sucrose in 1% concentration and glucose in a concentration of 0.1%. Citrate Utilization test was also done. This test determines the ability of microorganisms to utilize citrate. Some bacteria have the capacity to convert the salts of organic acids, for example, Sodium citrate to alkaline carbonates. Sodium citrate is one of the most important metabolite of Krebs cycle. Certain bacteria use citrate as the sole carbon source. Citrate utilization requires a specific membrane transport and citrate lyase activity. Citrate is converted to Oxalo acetic acid by citrate lyase and oxaloacetate decarboxylase activity will convert oxaloacetate to pyruvate with the release of carbon dioxide. The carbon dioxide reacts with sodium and water to form sodium carbonate. Urease test was also one of the biochemical tests looked in this practical. Urea is a nitrogen containing compound that is produced during the decarboxylation process of the amino acid arginine in the urea cycle. Certain bacteria produce the enzyme Urease during its metabolism process and that will break down the urea in the media and carbon dioxide: Some enteric bacteria produce the enzyme Urease, which splits the urea molecule into carbon dioxide and ammonia. The Urease test is useful in identifying the genera Proteus, Providentia and Monganella, which liberate this enzyme. (H2S) 2CO + 2H2O >>>>>>>>>> CO2 + H2O + 2NH3 SIM test stands for Hydrogen sulphide, Indole, Motility test, is a semi – solid media meant to test for hydrogen sulphide gas production and the metabolism of the amino acid tryptophan. This test is also solely known as the Indole test which looks for the presence or absence of tryptophanase enzyme production of the bacteria. If the enzyme is present, it will degrade the amino acid tryptophan in the media and will produce Indole, ammonia and pyruvic acid. Indole will react with Kovac’s reagent to produce a cherry red complex, which indicates a positive Indole test. The absence of red color is indicative of tryptophan hydrolysis due to the lack of tryptophanase enzyme. SIM agar may also be used to detect the presence of H2S production. Hydrogen sulphide is liberated as a result of the degradation of sulphur containing amino acids. A black precipitate forms due to the reaction of hydrogen sulphide with the iron salts in the medium. It can be said then that through biochemical tests performed in the laboratory, a more conclusive identification of the different enterobacteriaceae species can be done.
  4. 4. MATERIALS             Cultures A & B Bunsen burner Inoculating wire –straight wire Inoculating loop Urea agar Citrate agar TSI SIM Incubator Kovacs reagent Dropper Test tubes & racks PROCEDURE A. TSI (triple sugar iron) test for CHOs fermentation The inoculating loop was firstly sterilized by heating on a flame until it was red hot, the mouth part of the tube containing the agar was also sterilized by being passed over the Bunsen burner fame. The loop was then cooled and thereafter a single colony was picked using the loop, then slant was then streak followed by the butt. The lid was then placed over the test tube. This procedure was repeated for cultures A & B. B. SIM (H2S, Indole, Motility) tests In the same way as in A. above the inoculating loop was used. Firstly sterilized using the Bunsen burner flame and the test tube containing the media, mouth was also sterilized. A single colony was then picked using a sterilized loop. The butt was streaked. This was done to both cultures A & B. C. Citrate test A straight wire was first sterilized, cooled and then mouth of the tube containing the media was sterilized as well. A single colony was collected from the culture plate and then the slant of the citrate test sample was gently inoculated. This was done to both cultures A & B. 4) UREASE TEST
  5. 5. The straight wire was first sterilized by heating till red hot over a Bunsen burner flame. The mouth part of the test tube containing the urea agar was also sterilized by being passed over the flame. Thereafter, a single colony was picked using a cooled straight wire from the culture plate then the butt was stabbed and the wire was removed via the same path of entrance. The lid was then placed over the test tube. This was done for both culture A and B. RESULTS Table1: showing results of laboratory biochemical bacterial identification tests Colors of media before inoculation were as follows; TSI agar --- brown Citrate-----green SIM--------colorless Urea-------orange Profile Observation TSI A Positive CITRATE UREA Negative Negative  Yellow/Yellow (A/A)  H2S absent  Gas present Observation B Positive S Negative  Green= citrate utilized  Orange= Urease absent  H2S absent I Positive  Pink= Indole present Negative  Motility present Positive Positive Negative Positive  Black ppt.butt(K/A)  H2S present  Gas present  Blue= citrate utilized  Orange= Urease absent  H2S present SIM M Positive Culture A: E.coli Culture B: Salmonella  Colorless= Indole absent  Motility present
  6. 6. DISCUSSION The four biochemical tests were carried out in order to differentiate and identify enterobacteria and in this lab practical the bacteria identified was E.coli and Salmonella. In TSI test, culture A had an acidic slant and an acidic butt (A/A), indicating that there was glucose, sucrose and/or lactose fermentation. In this test tube, hydrogen sulphide was not produced butt there was the presence of gas. Culture B had an alkaline slant and acidic butt (K/A), which indicated that glucose was fermented anaerobiaclly and showed that there was the production of hydrogen sulphide and gas. In the citrate test, culture A tested negative for citrate utilization and culture B tested positive shown by the formation of the blue color. In the Urease test, culture A and B tested negative for urea production showing that these bacteria had no ability to produce Urease enzyme. The SIM test, three things were being tested for thus, hydrogen sulphide, Indole and motility. Culture A tested negative for hydrogen sulphide while culture B tested positive. For Indole test, culture A tested positive while culture B tested negative. Finally for Motility test both culture A and B tested positive.Kovac’s reagent is is an isomyl alcohol scientifically known as paradimethylamino benzoaldehyde Therefore, based on the total of these results, culture A and B were identified as E.coli and Salmonella respectively. CONCLUSION The cultures A and B of enterobacteriaceae were identified as E.coli and Salmonella respectively. REFERENCES 1) Ikram M. and Hill E. (1991), Microbiology for Veterinary Technicians, 1st Edition,American Veterinary Publications Incorporated, California. 2) Nester W.E et al (2004), Microbiology: A Human Perspective, 4th Edition, McGraw Hill Companies, New York. 3) http://www.scienceprofonline.com/microbiology/bacteria-colony-morphology-identificationunknown bacteria.html retrieved at 14:05 hrs, 24/11/2013.s

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