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BMSC207 Microbiology and Immunology

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Laboratory:  Identification Of Unknown Bacteria

The Aim Of This Laboratory Session is to enable you to:

  1. Understand how bacteria are characterized and classified.
  1. Identify an unknown bacterium.

General Introduction 

In microbiology, a system of classification must be available to allow the microbiologist to categorize and classify microorganisms for a wide variety of reasons, such as:

1)  Determination of pathogens responsible for infectious diseases.

2) Selection and isolation of strains of fermentative micro-organisms necessary for the industrial production of alcohols, solvents, vitamins, organic acids, antibiotics and industrial enzymes.

3) Isolation and development of suitable microbial strains necessary for the manufactures and the   enhancement of quality and flavour in certain food materials e.g., yoghurt, cheeses and milk products.

4)  Comparison of biochemical activities for taxonomic purposes.

The most important reference for bacterial taxonomy is Bergey's Manual, in which bacteria are grouped into numbered sections, according to the Gram stain reaction, cell shape, cell arrangements, oxygen requirements, motility and nutritional and metabolic properties.  

You will use provided bacteria as examples to identify an unknown bacterium. By using careful deduction and systematically compiling and analyzing information, you should be able to identify your bacterium.

Materials to be prepared:

  1. Bacteria culture plates.
  2. Catalase and oxidase test kits.
  3. Fermentation test tubes for glucose, mannitol and lactose.
  4. Citrate slob is optional.

A key to species of bacteria is provided on pages 44-46. The key is an example of a dichotomous classification system; that is, a population is repeatedly divided into two parts until a description identifies a single member.

To begin your identification, ascertain the purity of the culture you have been given. Avoid contamination of your unknown. Note growth characteristics and Gram stain appearance for clues about how to proceed.

After staining the unknown, many bacterial groups can be eliminated. Final determination of your unknown will depend on careful selection of the relevant biochemical tests e.g.

Carbohydrate fermentation test

Oxidase test

Catalase test

Materials – Per Pair

Culture of Bacteria A and Bacteria B on NA culture

All stains, reagents and media needed.

Procedure

Work in pairs. Each pair will identify the bacterium A and bacterium B

  • Note the morphologic and other characteristics of your unknown bacterium by observing its colonies on the agar plate.
  • Perform a Gram stain and note the colour and arrangement of your stained bacteria.
  • Refer to the two supplied tables (on pages 7,8 & 9) to determine which biochemical tests you will need to perform to make a final identification.  Do not be wasteful. Inoculate only what is needed. Do not perform unnecessary tests.

Discussion

Your report should be a formal report and the discussion should contain the rationale for arriving at your identification. Make sure to answer all below questions

Explain the tests used to identify the bacteria.  Plus answer the following question.

Which type of bacteria was it?

What is the characteristic of the identified bacteria?

Is that bacteria gram positive or gram negative?

Which Bacteria was bacteria B and which bacteria was bacteria A

Does examination of colonial morphology ( such as Catalase test, oxidase, carbohydrates fermentation, indole and urease tests )in itself yield sufficient information to allow identification of microorganisms?  Explain.

BMSC207 Microbiology and Immunology

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