Mannitol Salt Agar (MSA)

Mannitol salt agar contain high salt content making the growth selective for bacteria that can grow in high level of NaCl which is normally inhibitory to most other bacteria.

Mannitol Salt Agar is both selective and diffferential

Selective:
Mannitol salt have very high level salt content hence it only allow the growth of Staphylococci as most other bacteria are unable to withstand the high salt condition for growth.It allows the growth of Gram positive bacteria and inhibits the growth of Gram negative bacteria.

Differential:
MSA contains mannitol and indicator phenol red.
*Coagulase-positive Staphylococci will produce yellow colonies with yellow zone
*Coagulase-negative Staphylococci will produce small pink or red colonies with no colour change to the medium

http://images.google.com.sg/imgres?imgurl=http://micro.farfallas.com/msa.jpg&imgrefurl=http://micro.farfallas.com/index.php

Blood Agar

Blood Agar are used to isolate organisms and detect hemolytic activity.

Blood Agar are enriched, differential media.

β-hemolytic activity will show complete lysis of red blood cells surrounding the bacteria.
Example of bacterias are Streptococcus haemolyticus

α-hemolysis activity will only show partial lysis of hemoglobin and will appear green.
Example of bacterias are Streptococcus viridans

http://images.google.com.sg/imgres?imgurl=http://www.sheep101.info/Images/agarplates.jpg&imgrefurl=http://www.sheep101.info/bloodsheep.html

Catalase Test

What is catalase test?
Catalase is an enzyme that decomposes hydrogen peroxide into oxygen and water.This test is used to distinguish Gram-positive cocci,to differentiate between streptococci for staphylococci. To run this test we will need well isolated colonies of an 18-24 hours cultures, 3%hydrogen peroxide solution, clean glass slide, enoculating loop and a dropper.


How would we go about running this test?
1.transfer cells from centre of the isolated colonies onto the clean glass slide
2.add 2 drops of 3% hydrogen peroxide directly onto the smear
3.record the observation

http://ftp.ccccd.edu/dcain/CCCCD%20Micro/catalase_test.htm

What are the observations?
*Catalase-Positive will form gas bubbles rapidly
*Catalase-Negative will have no production of gas bubbles


Conclusion?
Member of the Staphylococcus are catalase-positive.
Members of the Streptococcus are catalase-negative.

Introduction to Selective medium & Differential medium

Selective Media:
· Isolates specific groups of bacteria
· Contains inhibitors which prevent the growth of unwanted bacteria, while allowing the growth of the desired bacteria

Differential Media:
· Distinguishes between biochemically and morphologically related groups of bacteria
· Incorporates chemicals that produce some characteristic change in the colonies or media around the colonies of specific types of bacteria within the group

MacConkey Agar

·Purpose: MacConkey Agar is used to isolate and differentiate members of the Enterobacteriaceae based on the ability to ferment lactose.

·MacConkey Agar is both selective and differential

i. Selective:
Ø Contains bile salts and crystal violet
Ø Inhibits growth of gram-positive bacteria
Ø Isolates gram-negative bacteria

ii. Differential:
Ø Contains lactose and neutral red
Ø Lactose fermenters produce acid, causing the colonies to turn red
Ø Non-lactose fermenters do not produce acid. Thus, colonies either remain their normal colour or the colour of the medium

·Examples of selective and differential MacConkey agar:

i. Selective:











https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEifA_dd_12HTAv2_R10mgzL4Q-BRbnTNLSHvXmvRW-ezfe0LMwNwEhv4lGWSpGP0QsrRgZrXqp-XXhmEAvjRNJIzta4CNxI44panv99Ms9ygxegeKV1kHABQPfxh70IQjnds4KTm3DqP8p7/s1600-h/MacConkey_agar.jpg


Gram-positive bacteria- red colonies
Gram-negative bacteria- colourless colonies

ii. Differential:













https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhQ_TFHYc2h1h031cuzklhUDWXGlWEGXcZ3FpvnBAAaeUFw290eXTDNlhWZyBCHI2cDPZ3D8JxvukJxamjoBl3RUiINEUK54_Oiz7l6fSJtOHaI5W7sjPX6TYGc_NxC35iLreATAz6QIvjr/s1600-h/MacConkey_agar_with_LF_and_LF_colonies.jpg

Lactose fermenters turn the colonies pink.
Colonies of the non-lactose fermenters are colourless.

Hektoen Enteric Agar

· Purpose: To isolate and differentiate Salmonella and Shigella species from other gram-negative enteric organisms.

· Contents of HE Agar: Dyes and bile salts.

· Uses of the contents in HE Agar: Inhibits growth of gram-positives and high concentrations of three sugars: lactose, sucrose and the plant glycoside salicin. Sugars that produce acid, changes the colour of the dyes. Acid-producers form orange or pink colonies and non-acid producers form greenish colonies.

· Examples of acid producers and non-acid producers:



















E. coli on Hektoen enteric agar
Bright yellow-orange colonies are formed

Salmonella enteric on Hektoen enteric agar

Greenish colonies are formed

Introduction to IMViC test

Bacteria can be differentiated from one another and can be achieved by means of the IMViC set of tests. I stands for the Indole test, the M represents Methyl red test, the V refers to the Voges-Proskauer reaction, and the C is for citrate test.

The Indole Test

The amino acid tryptophan is found in nearly all proteins.

Positive Indole: Bacteria that contain tryptophanase can hydrolyse tryptophan to its metabolic products(indole, pyruvic acid, ammonia)

Tryptophan ----Tryptophanase-----> Indole + Pyruvic Acid + Ammonia

The bacteria use the pyruvic acid and ammonia to satisfy nutritional needs; indole is not used and accumulates in the medium.
This will form a layer of red color on the surface of the medium when added with Kovac’s reagent due to the combination of aldehyde in the Kovac’s reagent and indole.

Indole + added Kovac’s reagent = Red color

Negative Indole: There would be no red color formation on the agar surface when added with Kovac’s solution.

Tryptophan ----No Tryptophanase-----> Tryptophan

Tryptophan + added Kovac’s reagent = no red color



http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/Johnson/images/INDOLE%20Enterobacter%20Escherichia%20coli%20fig1.jpg

The Methyl Red Test & Voges-Proskauer Test

The methyl-red(MR) and Voges-Proskauer(VP) tests are used to differentiate two major types of facultatively anaerobic enteric bacterial species based on their pattern of glucose metabolism.

Methyl Red Test
All enterics initially produce pyruvic acid from glucose metabolism.
Glucose -----Glucose Metabolism----> Pyruvic acid

Positive result: Some enterics subsequently use the mixed acid pathway to metabolize pyruvic acid to other acids(lactic, acetic, and formic acids).
Pyruvic acid -----Mixed Acid Pathways------> lactic, acetic, and formic acids
Many acids (pH 4.2) + added methyl red = red color
Bacteria are called methyl-red-positive.

Negative result: Other enterics subsequently use butylene glycol pathway to metabolize pyruvic acid to neutral end-products.
Pyruvic acid------Butylene Glycol Pathway------> neutral end-products
Neutral end-products (pH 6.0) + added methyl red = yellow color
Bacteria are called methyl-red-negative.

http://web.clark.edu/tkibota/images/MethylRed.gif

Voges-Proskauer Test
A test to distinguish between acetoin-producing enterics and enterics that do not produce acetoin. (Acetoin is otherwise known as acetylmethylcarbinol) Enterics that subsequently produce neutral end-products from pyruvic acid are detected with the Voges-Proskauer test.

Positive result:
Glucose ------Glucose Metabolism-------> Pyruvic Acid
Pyruvic acid ---------------> Acetoin
Acetoin + added alpha-naphthol + added KOH = red color

Negative result:
Glucose ------Glucose Metabolism-------> Pyruvic Acid
Pyruvic acid -----------------> No Acetoin
No acetoin + added alpha-naphthol + added KOH = copper color

http://www.mc.maricopa.edu/~johnson/labtools/Dbiochem/vp.jpg

Citrate Test

Citrate is an organic molecule that can be utilized by some bacteria but not by others.

The Simmon's citrate agar plates or slants:

- Determine whether an organism produces citrase
- Content: citrate as the only carbon source and bromthymol blue (pH indicator)

Positive test:

Bacteria with the enzyme citrase metabolize citrate to produce alkaline end-products that raise the pH of the medium to 7.6, causing the bromthymol blue to turn blue.

Citrate(green) -------Citrase------> oxaloacetic acid + acetic acid

Oxaloacetic acid --------------------> Pyruvic acid + Carbon dioxide
Carbon dioxide + Sodium ions ----------> Sodium Carbonate(pH increases)

[Blue and growth]

Negative test:

Citrate(green) -------No citrase-----> Citrate(pH unchanged)
[Green and no growth]

http://www.medschool.lsuhsc.edu/Microbiology/DMIP/citrate.jpg