METABOLIC ACTIVITIES OF MICROORGANISMS

Group 1Bernas, Boris et.al

INTRODUCTION

Microorganisms grow and multiply by using raw materials found in their environment. The available

nutrients in the environment may consist of simple to complex molecules. They can oxidize these nutrient to obtain energy and modify then to build precursors for

the synthesis of necessary cellular components. Microorganisms utilize nutrients in a variety of ways and the metabolism often produces by-products that can be used in the differentiation of microorganisms.

The production of the visible pigments is often used in distinguishing groups of microorganisms particularly

bacteria.

Objectives• Perform the biochemical tests used in

identification of bacteria• Explain the principle behind the production

of metabolic by-products• Emphasize the importance of fermentation

reactions in the identification of bacteria• Become aware on the importance of

enzyme production by bacteria

Materials• inoculating loop• Clean test tube• Disinfectant• 24 hr-old broth cultures of• E.coli• P.vulgaris• E.aerogenes• S.epidermidis• M.luteus• S.aureus• P.aeruginosa• S.Faecalis

Culture media Basal medium

› Glucose› Maltose› Sucrose› Lactose› Mannitol

• Reagents– 10% ferric chloride– Methyl red– 40%KOH– 5% alpha naphthol– Oxidase reagent– Catalase reagent– Kovac’s reagent– Methyl red voges proskauer broth (MRVP)– SIM– LDC– DC– PA– SCA– CUA– NA

Results and Discussion

Bacteria BMglu BMmal BMlac BMsuc BMman

E. coli AG N AG N A

M. luteus N N N V N

P. vulgaris AG AG N AG A

S. aureus A N A A A

Unknown A N N N N

Table 1.1

Fermentation Test

Tube 1: Negative acid /Negative gasTube 2A: Must incubate longer (ambiguous

result)Tube 2B: Positive acid /Negative gasTube 3A: Positive acid/ Positive gas

Results of Fermentation test

Results of Fermentation test

Results of Fermentation test

Results of Fermentation test

Principle of Fermentation of sugar

To determine the ability of an organism to ferment a specific carbohydrate (sugar) incorporated in a medium producing acid or acid with gas.

Type of fermentation

Explain the type of fermentation of the unknown.

-the unknown bacteria is a bacteria that belongs to the family of Enterobacteriaceae because that bacteria ferments glucose. One characteristic of the family Enterobactericeae is they are glucose fermenters.

Methyl red and Voges Proskauer

Bacteria Methyl Red Voges Proskauer

E.coli + -

E. aerogenes - +

Unknown - +

Table 1.2

Methyl red (MR)

this test detects the production of sufficient acid during fermentation of glucose by bacteria and sustained maintenance of a pH below 4.5.

MR—tests for acid end products from glucose fermentation.

Methyl Red (MR)

Tube A.  Methyl red negative

Tube B.  Methyl red positivePositive control: E. coli, Listeria monocytogenes

Negative control: Klebsiella, Enterobacter spp.

E. Coli group (+)

Ferments glucose Produce acid by & end products Low pH (4.5 and below) Methyl red turns to red

Enterobacter klebsiella group (-)

Ferments glucose Produce non-acidic by & end products

(alcohol, acetion & acetyl methyl carbinol

High pH 6.2 Methyl red turns to yellow

Voges Proskauer

This test depends on the production of acetyl methyl carbinol (acetoin) from pyruvic acid in the media. In the presence of alkali and atmospheric oxygen, acetoin is oxidized to diacetyl which reacts with α-naphthol to give a red color.

VP—tests for acetoin production from glucose fermentation.

Voges Proskauer

Tube A.  Voges-Proskauer positive

Tube B.  Voges-Proskauer negative

Positive control: Klebsiella sp., Enterobacter sp., Staphylococcus.

Negative control: E. coli, Micrococcus.

Based on the table, is it possible to have both MR and VP positive?

Based on the table, it is not possible to have both MR and VP. It is because these two tests aims to differentiate one species of microbes from another by their end-products which are acetoin for VP and acid for MR.

Catalase and Oxidase test for bacteria

Bacteria Catalase Oxidase

E.coli + + - -

P. flourescens + + -

S. epidermidis + + -

S. faecalis - -

Unknown + -

Table 1.3

Principle of Catalase and Oxidase

certain bacteria have an enzyme catalase which acts on hydrogen peroxide to release oxygen.

Positive test: Immediate bubbling easily observed

Negative test: No bubbling

Catalase

Positive control: Staphylococcus, Micrococcus, all members of Enterobacteriaceae except Shigella dysentriae.Negative control: Shigella dysenteriae typeI, Streptococcus, Clostridium.

Catalase

Bubbles appear indicating a positive result

Oxidase

To determine the presence of an enzyme cytochrome oxidase which catalyses the oxidation of reduced cytochrome by molecular oxygen.

Positive: deep purple within 10 secs.Negative: – No color change

Oxidase

Positive control: Pseudomonas sp., Vibrio sp., Alcaligenes sp.Negative control: All members of Enterobacteriaceae

Utilization of amino acids of the test bacteria

Bacteria Indole Production

Sulfide Production

Lysine Production

Phenylalanine Production

E.coli - + - - - + - -

E. aerogenes

- - - - - - -

C. freundii - + - - - - -

P. vulgaris + + + + - - + +

unknown - + + -

Indole

Testing for indole production is important in the identification of Enterobacteria. Most strains of E. coli, P. vulgaris, Povidencia species break down the amino acid tryptophan with release of indole.

The test organism is cultured in a medium which contains tryptophan. Indole production is detected by Kovac’s or Ehrlich reagent which contains 4(p)-dimethylaminobenzaldehyde. This reacts with the indole to produce a red colored compound.

Indole

Positive indole control: E. coli.Negative indole control: Klebsiella

pneumoniae, Enterobacter aerogenes.

Sulfide Production

This test is used to help differentiate species of the family Enterobacteriaceae. This test is used to determine the ability to reduce sulfur into H2S.

Sulfide production

H2S will react with the iron or ferrous sulfate and produce a black precipitate. A positive result has a black precipitate present and a negative result has no black precipitate

Citrate and Urea Utilization of test bacteria

Bacteria Citrate Urea

E. coli - - - -

E.aerogenes + + - -

P. vulgaris - +/- + +

Unknown - -

Urea This test is done to determine a bacteria’s

ability to hydrolyze urea to make ammonia using the enzyme urease.

Urea broth is a yellow-orange color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made.

Bacteria Indole production

Methyl red Voges Proskauer

Citrate concentration

E.coli + + - -

E. aerogenes

- - + -

IMViC reaction of E. coli and E.aerogenes

Identity of unknown bacteria

Salmonella typhii

Conclusion and Recommendation

Different tests are needed to be conducted for us to better understand to what certain chemicals and enzymes they might react for easier determination of the different reactivity and identity of the bacteria.

Based on the unknown bacteria that was assigned to us we have identified it as Salmonella typhii

Conclusion and Recommendation

We recommend that practicing of these tests regularly will ensure the accuracy and preciseness of the tests being conducted in the perspective of being a microbiologist.