Biochemical tests- MICROBIOLOGY

Oxygen is sometimes toxic.

Small amounts of superoxide free radicals are formed during the normal respiration of organisms that use oxygen as the final electron acceptor.

Obligate anaerobes from some oxygen free radicals that are toxic to the cell. Hence, if bacteria wants to grow in oxygen environment, enzymes like catalase and superoxidase dismutase must be present for neutralization of the toxic form of oxygen(oxygen radical)

aerobes-anaerobes

During normal aerobic respiration, hydrogen ions are produced and have to be removed by bacterial cell. The electron transport system (ETS)  in cellular respiration (a part of glycolysis) involves these H+ ions and combines them with oxygen to form water. Water is harmless. Energy is given off and stored in the form of Adenosine Triphosphate.

What is toxic is Hydrogen Peroxide that is formed by the cytochromes in ETS. Water being harmless is not required to be removed by the bacteria. So, what is harmful to bacteria cell that requires it to be removed instantly??

answer: H2O2.

Functions of catalase

Protects bacteria from toxic hydrogen peroxide (H2O2) accumulation, which can occur during aerobic metabolism. If hydrogen peroxide accumulates, it becomes toxic to the organism.

Since Catalase breaks H2O2 down into water and O2, the presence of oxygen can be characterized by bubbles which indicates a (+) result.

What bacteria could mostly likely be detected?

most aerobic organism make catalase. catalase +

http://student.ccbcmd.edu/courses/bio141/labmanua/lab8/catstaph.html

Most aerobic organsims will display (+) results.

e.g. Staphyloccocus aureus.

Some anaerobic organisms will display (-) results, indicating that they do not produce catalase to prevent oxygen accumulation. Why?

Because since oxygen is totally not used for survival of these organisms, they do not have the ability to produce catalase.

e.g. Clostridium, Lactobacillus, Streptococcus

glycolysis

Fermentation can occur in the presence of oxygen or absence of oxygen.

If bacteria utilises carbohydrates for nutrients, there may be 2 end products, a gas and acid.  The substrate formed from the metabolism of carbohydrate is either glucose or lactose.

Even if bacteria releases enzymes that enable to use carbohydrates through fermentation and oxidation, gas may or may not be produced.

FERMENTATION is noted by acid production which can be observed by a colour change in Durham tubes aka  carbohydrate fermentation tube.

Phenol Red indicator is red in neutral or alkaline solution

If acid is present(+), phenol red changes from red –> yellow

If there is a small “space” at the top of the small tube, it means that gas is trapped in the small  inverted tube inside the bigger tube. Therefore, gas is produced from the breakdown of carbohydrates.

durham fermenation tube with gas produced

Alfred.E.Brown. (2007).  Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.

reaction in fermentation tubes

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.

Look at the 1st picture above, why is it red if gas is produced and it just means fermentation has occured?

Due to prolonged incubation periods(more than 24h), bacteria will begin to grow oxidatively(oxygen dependent) on the peptone contents of the fermentation medium after using up the carbohydrate contents, causing the neutralization of phenol red indicator and turing it red due to NH3 production.

table of durham fermentation

The only organism that has the ability to break down carbohydrate into glucose and lactose is E.coli and it also has the ability to produce gas during fermentation.

For P. aerguinosa, it displayed negative results for all, which means it can’t break down carbohydrate into glucose & lactose.

Another test which is the MRVP test is able to differentiate between organisms that produce large amounts of acid and organisms that only produce neutral content(acetoin)

M-Methyl Red. VP- Voges -Proskauer. Methyl Red is different from Phenol Red that we mention earlier. It has a pH of 4.4-6. Hence, it changes colour at this range.

For MR test,  if  organic acid is produced here, the (+) result is just a no change in colour of Methyl Red. RED remains.

if neutral acetoin is produced here, the (-) result is a change in colour from RED to YELLOW because at pH 7, the indicator will change its colour.Methyl red test

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.

To test for the presence of acetoin, we use VP test where potassium hydroxide and α naphthol are added.

The upper of the medium will turn red. (+) If the medium turns light brown, it is a (-) result.

Please note that production of acetoin is also affected by the duration of incubation, hence, false negative results may be observed.

We can use Koser’s citrate or Simmon’s citrate to test for the ability of bacteria to ferment citrate. When citric acid or sodium citrate is in solution, it loses a proton or sodium ion to form a citrate ion. Bacteria with citrate lyase can break down citrate to form pyruvate. Pyruvate can be further reduced in fermentation.

Purpose of performing the citrate test

  • Tests for the ability of bacteria to convert citrate (an intermediate of the Kreb’s cycle) into oxaloacetate (another intermediate of the Kreb’s cycle)

Contents  of Simmon citrate include

  • sodium citrate as the carbon source
  • monoammonium phosphate as the nitrogen source
  • and bromthymol blue indicator that changes to blue when medium turns alkaline, which means  (+) result.

Why alkaline means citrate is utilised by the bacteria?

When bacteria uses citrate(carbon) and ammonium(nitrogen), medium turns alkaline as ammonia is produced from ammonium.

citrate test

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.

Citrobacter, Enterobacter will display (+) result while E. coli & Klebsiella will display (-) result.

enterbacteriaceae family

After knowing that some bacteria utilises citrate, we can also find out if other products are formed during metabolism. For example, Hydrogen Sulphide.
hydrogen sulphide prod

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.

How do we determine if type of metabolism is fermentative or oxidative?ecoli, eaerogenes (controls included) OF test

we will test with an glucose  OF oxidative- fermentative agar. If agar turns yellow, acid is produced. If agar turns green, no acid is produced. Motility of the bacteria can also be determined by the presence of turbidity (cloudiness)

OXIDATIVE metabolism may or may not cause an acid to be produced for aerobic conditions. No acid will be produced for anaerobic conditions.

FERMENTATIVE metabolism will cause an acid to be produced for aerboic and anaerobic conditions.

OF test(yellow-acid, green-no acid)

Alfred.E.Brown. (2007). Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.

table of oxidation and fermentation

UREASE breaks down urea into ammonia (NH3) & carbon dioxide (CO2).

 urease test

Alfred.E.Brown. (2007).  Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.

Properties of urea medium

Since there’s phenol red pH indicator, pH indicator changes from yellow to bright pink if NH3 is produced.

At more than pH 6.8, the colour change represents a (+) result

Proteus vulagris is one bacteria that produces urease to break down urea.

Tryptophan is degraded by tryptophanase into indole, ammonia, pyruvic acid. Pyruvic acid is then involved in metabolic pathway so that ATP energy for bacterial cell is generated.  While other products like NH3 and Pyruvate is metabolised, indole is not. Hence, it stays in the medium.

Upon addition of Kovac’s reagent, deep red ring at the top of the agar/ broth  is formed when Kovac’s reagent reacts with indole. This is the (+) result.

E.coli is one of the organism that can display (+) result since exoenzyme tryptophanase is produced.

tryptophan hydrolysis

Alfred.E.Brown. (2007).  Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.

Properties of Tryptic Soy Broth/Agar

-tryptone is one of the contents of TSB/ TSA and is derived from casein.(recall protein from protein hydrolysis)

Lipolysis

Posted on: July 25, 2009

Lipolysis is carried out by LIPASES.

Triglycerides(big molecules derived from lipids) are broken down into fatty acids & glycerol

If pH  of medium, used for testing whether lipase is present to break down lipids,  is lowered. It indicates acidic product (fatty acid) is formed.

Trigylceride used here is tributyrin that can be found in medium Spirit Blue Agar.

There are 2 indicators for (+) result.

  1. dark blue precipitate(tributyrin is completely broken down into fatty acids)  OR
  2. oil droplets (when tributyrin is not completely broken down into fatty acids)

Organism responsible for exhibiting (+) result is Staphyloccocus aureus.

fat hydrolysis

Alfred.E.Brown. (2007).Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.

Properties of Spirit Blue Agar

Contents- tributyrin (simple animal triglyceride)

Tributyrin acts as a substrate for exoenzyme lipase.

Proteolysis is carried out by PROTEASE.

In the following posts we will mention

- bacteria’s ability to hydrolyse casein, gelatin

- urea hydrolysis by detecting presence of urease(refer to post on nitrogen metabolism- urea hydrolysis)

-hydrogen sulphide production

skim milk (+)skim milk(-)
Casein(a protein) is broken down by protease into peptones and amino acids.

During the degradation process, polypeptide bonds are broken.

Once  the bonds are broken, amino acids are produced. A clear zone surrounding streak line of agar indicates a (+) result.

Organism that gives (+) result is Bacillus subtilis, Clostridium spieces. (-) results are indicative that organismdoes not cause a clear zone. an example of organism displaying (-) result is  Escherichia coli.casein hydrolysis

Alfred.E.Brown. (2007). Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.

Properties of skim milk agar

  • Skim milk agar contents- casein, lactose and other nutrients, which support growth of lactobacilli.
  • Gives the white colour to milk.

As seen from the above, we can infer if bacteria present in milk  is able to break down the casein in milk, milk may have abnormal particles that are not white in colour.

Application of this biochemical this is in food testing.

Another protein commonly found in food products is gelatin.

It is broken down by gelatinase into smaller polypeptides, peptones and amino acids that can cross the cell membrane and be utilised by the organism.
Property of Gelatin agar

INTERESTING to note: when gelatin is broken down via hydrolysis, it cannot solidify anymore, the areas of solid gelatin media where the organsim grows, will turn into liquid.  Even if you refrigerate this medium, the media will remains liquid.


gelatin hydrolysis

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.

Hence, a (+) result is indicated by the liquid state of gelatin. Bacillus subtilis is able to produce proteolytic exoenzyme gelatinase to give the (+) result.

Why is gelatin not used widely as a selective media for isolating bacteria? (hint: what is gelatin agar unique property?)

Most bacteria do not contain enzymes that liquefy gelatin.  Hence, it is not uselfy for isolating microbes for bacterial identification.

Decomposition of amino acid “cysteine” is detected by the formation of ferrous sulphide when Hydrogen Sulphide is release.

Why? Some bacteria have the ability to give off  H2S from sulphur containing amino acids after proteins are broken down into amino acids by enzymes.

When H2S is produced, sulfide ion reacts with the metal salt to product a black precipitate(+) result.


triple sugar iron agar

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education.

LOOK AT TUBE 3. the black ppt indicates that Hydrogen Sulphide was produced. (+). Bacteria that produces (+) are Citrobacter, Salmonella.

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