BIO 2440: Principles of Microbiology

7. Differential and Selective Media and Biochemical Tests

5.1: Background

Differential and selective media are used in microbiology to encourage the growth of some
bacteria but not others, as well as to help tell species apart based on certain growth
requirements. You may recall from Lab 1, which discussed culture media, that selective media
are those which only allow for the growth of certain bacteria and not others, and that differential
media allow microbiologists to tell two species apart based on some visible qualitative change.

Media may be both differential and selective-- for example, mannitol salt agar is selective in that
it only allows for the growth of Staphylococcus species, and differential in that a colour change
is observed when the bacteria that are able to grow on it also ferment the mannitol substrate in
the medium. Following are a number of differential and selective media you will use in the lab.
Each shows a list of ingredients, and the selective and differential ingredients are italicised for
your benefit. At the end of this chapter are also described certain biochemical tests which are
also used to differentiate bacterial species, but do not require the use of a growth culture.

5.2: Bile Esculin Agar

Bile esculin agar (BEA) is used to differentiate members of the genus Enterococcus (formerly
known as Group D streptococci) from those of the genus Streptococcus. Enterococci are able to
hydrolyse esculin, while streptococci cannot, and therefore produce a positive result.1

Ingredients

Beef extract, 11g

Enzymatic digest of gelatine, 34.5g
Esculin, 1g: differential ingredient
Oxbile, 2g: selective ingredient
Ferric ammonium citrate, 0.5g: indicator
Agar, 15g

Esculin, a glycoside occurring in trees in the genus Aesculus, may be hydrolysed into useable
glucose and esculetin using the enzyme esculinase.2
Oxbile is bile harvested from cattle and prevents the growth of certain Gram positive species on
BEA, as most Gram positive species lack certain components which decrease the cellular
envelope’s permeability.3

Ferric ammonium citrate is a water-soluble iron salt which forms a dark brown to black
precipitate with esculetin.4 This colour change indicates a positive result, since esculetin can
only be present in the medium if the bacteria is capable of esculin hydrolysis.

Use

BEA is often made in agar slants, but may be made in plates as well. Culture is inoculated with
a loop on the surface of the agar and incubated for 48 hours at 37ºC. If a plate is checked at 24
hours, black precipitate may not yet have formed and a false negative may be reported.5

Interpretation

Dark brown to black precipitate formed by a complex between esculetin and ferric ammonium
citrate will appear under growth of bacteria that is able to hydrolyse esculin into esculetin and
glucose. This blackening of the usually light tan agar is considered a positive result.1

1. Lindell, S. S. & Quinn, P. (1975). Use of bile-esculin agar for rapid differentiation of Enterobacteriaceae. Journal of
Clinical Microbiology 1(5), 440-443.
2. Trepeta, R. W. & Edberg, S. C. (1987). Esculinase (beta-glucosidase) for the rapid estimation of activity in bacteria
utilising a hydrolyzable substrate, p-nitrophenyl-beta-D-glucopyranoside. Antonie van Leeuwenhoek 53(4), 273-277.
3. Cremers, C. M., Knoefler, D., Vitvitsky, V., Banerjee, R., & Jakob, U. (2014). Bile salts act as effective protein-unfolding
agents and instigators of disulfide stress in vivo. Proceedings of the National Academy of Sciences of the United States of
America 111(16), E1610-E1619.
4. Qadri, S. M., DeSilva, M. I., & Zubairi, S. (1080). Rapid test for determination of esculin hydrolysis. Journal of Clinical
Microbiology 12(3), 472-474.
5. Chuard, C. & Reller, L. B. (1998). Bile-esculin test for presumptive identification of enterococci and streptococci: effects of
bile concentration, inoculation technique, and incubation time. Journal of Clinical Microbiology 36(4), 1135-1136.
5.3: Blood Agar

Blood agar is an enriched and differential complex medium which comprises a nutrient-rich base
medium with animal blood added to assess a bacterial species’ cytotoxicity.

Ingredients

Enzymatic digest of casein, 15g

Enzymatic digest of animal tissue, 4g
Yeast extract, 2g
Corn starch, 1g
Sodium chloride, 5g
Agar, 14g
5% sheep’s blood: differential

Sheep’s blood, or that of another animal, is added to an enriched agar base designed to
cultivate fastidious bacteria, in order to differentiate bacterial species based on their haemolytic
effects on the erythrocytes in that animal blood. α-haemolysis (alpha-haemolysis) is an
incomplete haemolysis resulting from the production of hydrogen peroxide by the bacteria,
which oxidises haemoglobin to methaemoglobin. β-haemolysis is complete haemolysis of blood
cells in the agar, caused by the production of exotoxins called haemolysins, which completely
lyse erythrocytes. γ-haemolysis is a term used to describe the absence of haemolysis.1

Use

Blood agar may be used to analyse the pathogenicity of clinically significant bacteria. Because
those species which lyse cells, including erythrocytes, pose a unique threat to humans and
animals, blood agar may be used to find those species in a mixed environmental sample which
pose the greatest health risk to human populations.2

Interpretation

α-haemolysis will leave a greenish zone around bacterial growth on a blood agar plate where
the haemoglobin in the blood has been oxidised. β-haemolysis leaves a complete zone of
clearance around bacterial growth. γ-haemolysis, or non-haemolysis, will leave no colour
change or zone of clearance underneath bacterial growth.
 1. Hebert, G. A. & Hancock, G. A. (1985). Synergistic hemolysis exhibited by species of staphylococci. Journal of Clinical
Microbiology 22(3), 409-415.
2. Payment, P., Coffin, E., & Paquette, G. (1994). Blood agar to detect virulence factors in tap water heterotrophic bacteria.
Applied and Environmental Microbiology 60(4), 1179-1183.

5.4: Eosin Methylene Blue Agar

Eosin methylene blue agar (EMB) is used to differentiate Gram negative coliforms (those Gram
negative bacilli which can ferment lactose and are non-spore forming, e.g. Escherichia and
Klebsiella species) from non-coliforms (those which cannot ferment lactose, e.g. Salmonella and
Serratia species). Differentiating coliforms from non-coliforms is especially significant for testing
the safety and purity of public water supplies.1

Ingredients

Enzymatic digest of gelatine, 10g

Lactose, 10g: differential ingredient
Dipotassium phosphate, 2g
Eosin Y, 0.4g: selective ingredient; indicator
Methylene blue, 0.065g: selective ingredient; indicator
Agar, 15g

The dyes eosin Y and methylene blue are precipitated out in the presence of acidic metabolic
byproducts produced by lactose fermenters.2 This causes growth of lactose fermenters to
appear dark in colour.

These dyes are also toxic to Gram positive bacteria, inhibiting their growth and making EMB a
selective medium.3

Use
EMB agar is used to differentiate Gram negatives based on their ability to ferment lactose.
Culture is inoculated with a loop on the surface of the agar and incubated at 37ºC. Coliform
bacteria should be easily identified after 24 hours of incubation at this temperature.4

Interpretation

If darkening of the bacterial growth occurs, it may be concluded that that bacteria is able to
ferment lactose. Darker growth indicates stronger lactose fermentation. Escherichia coli,
uniquely, displays dark growth with a characteristic green metallic sheen due to its vigorous
fermentation of lactose.4 No darkening of growth or a very slight pink tinge to growth shows very
weak to no lactose fermentation.

The streak at the top of the plate shows E. coli growth with characteristic green metallic sheen
caused by strong lactose fermentation yielding highly acidic metabolic byproducts (coliform).
The streak at bottom shows pink mucoid growth, implying some acidic byproduct production,
likely through weak lactose fermentation (possible coliform). The streak to the right shows dark
purple growth characteristic of moderate lactose fermentation (probable coliform). The streak at
the left shows no precipitation of dye and therefore is not fermenting lactose (Gram negative
non-coliform).

1. Johnson, T. J. (2011). Impacts of fecal bacteria on human and animal health--pathogens and virulence genes. In M. J.
Sadowsky & R. L. Whitman (Eds.), The Fecal Bacteria (135-164). Washington, DC: the American Society for
Microbiology.
2. Wanger, A., Chavez, V., Huang, R. S. P., Wahed, A., Actor, J. K., & Dasgupta, A. (2017). Media for the Clinical
Microbiology Laboratory. In A. Wanger et al. (Eds.), Microbiology and Molecular Diagnosis in Pathology: a
Comprehensive Review for Board Preparation, Certification and Clinical Practice (51-60). Cambridge, MA: Elsevier.
3. Churchman, J. W. (1913). The selective bactericidal action of methylene-blue. Journal of Experimental Medicine 18(2),
187-189.
4. Leininger, D. J., Roberson, J. R., & Elvinger, Francois. (2001). Eosin methylene blue agar to differentiate Escherichia coli
from other gram-negative mastitis pathogens. Journal of Veterinary Diagnostic Investigation 13, 273-275.
5.5: Mannitol Salt Agar

Mannitol salt agar (MSA) contains a high concentration of salt (7.5%) in order to select for
growth of halophilic Gram positive cocci (Staphylococcus species and Micrococcaceae) and is
differential between mannitol-fermenting species (usually coagulase positive Staphylococci) and
non-mannitol-fermenting species (usually coagulase negative Staphylococci) based on the
reaction of byproducts with the phenol red indicator.1

Ingredients

Enzymatic digest of casein, 5g

Enzymatic digest of animal tissue, 5g
Beef extract, 1g
D-mannitol, 10g: differential ingredient
Sodium chloride, 75g: selective ingredient
Phenol red, 0.025g: indicator
Agar, 15g

High salt concentrations inhibit the growth of Gram negative bacteria, allowing for the selection
of Gram positive cocci from mixed cultures.2 Salt affects the turgor pressure of the cell, and
those bacteria which are unable to withstand high salt concentration are subjected to
hyperosmotic shock and undergo plasmolysis, killing the cells.3

D-mannitol is a sugar alcohol which is generally only utilised by coagulase-positive

Staphylococcus species, such as S. aureus and S. indermedius.4 There are, however,
exceptions to this rule, as certain methicillin-resistant S. aureus (MRSA) strains do not utilise
mannitol and certain coagulase negative species, such as S. caprae, still produce yellow
colonies on MSA.5

Phenol red (phenylsulphonphthalein) is a pH indicator which changes colour from red at a

neutral pH (~7) to yellow below pH 6.8 (acidic) or bright pink above pH 8.2 (alkaline).6

Use

Culture is inoculated with a loop on the surface of the agar and incubated for 24 hours at 37ºC.

Interpretation

The acidic metabolic byproducts produced by species able to ferment mannitol on MSA turn the
phenol red in the medium yellow. Species unable to ferment mannitol will not produce acidic
byproducts and therefore will leave the medium red to pink in colour.7
 1. Anderson, C., Johnson, T. R., Case, C. L., Cappuccino, J. G., & Sherman, N. (2015). Great Adventures in the
Microbiology Laboratory. Pearson.
2. Hill, J. H. & White, E. C. (1929). Sodium chloride media for the separation of certain Gram positive cocci from Gram
negative bacilli. Journal of Bacteriology 18(1), 43-57.
3. Wijnker, J. J., Koop, G., & Lipman, L. J. A. (2006). Antimicrobial properties of salt (NaCl) used for the preservation of
natural casings. Food Microbiology 23(7), 657-662.
4. Hanselman, B. A., Kruth, S. A., Rousseau, J., & Weese, J. S. (2009). Coagulase positive staphylococcal colonisation of
humans and their household pets. The Canadian Veterinary Journal 50(9), 954-958.
5. Thakur, P., Nayar, C., Tak, V., & Saigal, K. (2017). Mannitol-fermenting and tube coagulase-negative Staphylococcal
isolates: unraveling the diagnostic dilemma. Journal of Laboratory Physicians 9(1), 65-66.
6. National Center for Biotechnology Information. PubChem Compound Database; CID=4766,
https://pubchem.ncbi.nlm.nih.gov/compound/4766 (accessed 14 Mar. 2019).
7. Wertheim, H., Verbrugh, H. A., van Pelt, C., de Man, P., van Belkum., A., & Vos, M. C. (2001). Improved detection of
methicillin-resistant Staphylococcus aureus using phenyl mannitol broth containing aztreonam and ceftizoxime. Journal of
Clinical Microbiology 39(7), 2660-2662.

5.6: Simmons’ Citrate Agar

Simmons’ citrate agar is used to differentiate faecal coliforms from non-faecal coliforms based
on their ability to utilise citrate as a sole source of carbon and ammonium dihydrogen phosphate
as a sole source of nitrogen.

Ingredients

Ammonium dihydrogen phosphate, 1g: differential ingredient

Dipotassium phosphate, 1g
Sodium chloride, 5g
Sodium citrate, 2g: differential ingredient
Magnesium sulphate, 0.2g
Bromothymol blue, 0.08g: indicator
Agar, 15g

Ammonium dihydrogen phosphate is the only source of nitrogen found in the medium, and must
be metabolised by the organism in order to successfully grow on the medium. Metabolism of
ammonium dihydrogen phosphate produces ammonia as a byproduct, and this changes the pH
of the medium, making it more alkaline and causing the colour change.2

Sodium citrate may be metabolised using the citric acid cycle, but only if the bacteria have the
enzyme citrate permease, which imports citrate across the bacterial membrane alongside
certain cations.4

Bromothymol blue (3,3’-dibromothymolsulfonphthalein) is a pH indicator which appears blue

above pH 7.6, green at neutral pH (6.0-7.6), yellow at acidic pH (below 6.0), and pink at
extremely acidic pH (~0).3

Use

Culture is inoculated with a loop on the surface of the agar slant and incubated for 48 hours at
37ºC.

Interpretation

A positive reaction indicates an increase in alkalinity as a result of ammonium dihydrogen

phosphate and citrate utilisation, and growth of the cultured species. No growth, and therefore
an absence of colour change, indicates that the bacteria cannot use citrate as a sole carbon
source and is a probable coliform. Growth without a colour change may also be interpreted as a
negative result.

1. Simmons, J. S. (1926). A culture medium for differentiating organisms of typhoid-colon aerogenes groups and for isolation
of certain fungi. Journal of Infectious Diseases 39(3), 209-214.
2. Koser, S. A. (1923). Utilisation of the salts of organic acids by the colon-aerogenes group. Journal of Bacteriology 8(5),
493-520.
3. De Meyer, T. (2014). Substituent effects on absorption spectra of pH indicators: An experimental and computational study
of sulfonphthaleine dyes. Dyes and Pigments 102, 241-250
4. Warner, J. B. & Lolkema, J. S. (2002). Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+-citrate
transporter CitM. Microbiology 148(11), 3405-3412.
5.7: SIM Medium

The sulphide, indole, motility (SIM) medium is used to differentiate enteric bacterial species
based on the formation of indole, motility, and the production of sulfide. Formation of indole is
catalysed by tryptophanase, a lyase enzyme found in many species found in the intestines.1
Indole is a quorum-sensing signal molecule which aids in bacterial virulence through biofilm
formation and antibiotic tolerance.2

Ingredients

Pancreatic digest of casein, 20g: differential ingredient

Peptic digest of animal tissue, 6.1g
Ferrous ammonium sulphate, 0.2g
Sodium thiosulphate, 0.2g
Agar, 3.5g

Pancreatic digest of casein contains amino acids and is especially rich in tryptophan, which
certain bacterial species are able to break down into indole, pyruvate, and ammonia using the
enzyme tryptophanase.1

Use

Bacterial culture is picked up on a sterile inoculating needle, which must then be stabbed two
thirds of the way to the bottom of the medium. Incubate 48 hours at 37ºC.

Interpretation

To detect the production of indole, add three to four drops of Kovacs’ reagent. The para-
dimethylaminobenzaldehyde (DMAB) will react with indole, if present, to form a bright cherry-red
layer.2
 1. Nuidate, T., Tansila, N., Panthong, K., & Vuddhakul, V. (2016). Characterisation of tryptophanase from Vibrio cholerae
O1. Procedia Chemistry 18, 185-189.
2. Zhang, S., Zhang, W., Liu, N., Song, T., Liu, H., Zhao, X., Xu, W., & Li, C. (2017). Indole reduces the expression of
virulence related genes in Vibrio splendidus pathogenic to sea cucumber Apostichopus japonicus. Microbial Pathogenesis
111, 168-173.

5.8: Glucose Fermentation Test (Phenol Red Fermentation Broth)

Phenol red fermentation broth (PRFB) is used to determine whether a bacterial species is able
to ferment certain carbohydrates. Any carbohydrate, such as glucose, sucrose, fructose, or
lactose, may be used.

Ingredients

Pancreatic digest of casein, 10g

Sodium chloride, 5g
Phenol red, 18mg: indicator
Single carbohydrate, 5-10g: differential ingredient

Phenol red (phenylsulphonphthalein) is a pH indicator which changes colour from red at a

neutral pH (~7) to yellow below pH 6.8 (acidic) or bright pink above pH 8.2 (alkaline).2

Addition of a single carbohydrate allows for determination of whether the species inoculated in
the medium is able to ferment that carbohydrate.

Use

Inoculate the medium by placing a small amount of bacterial culture on the wall of the test tube
and mix it into the broth. Incubate for 48 hours at 37ºC.

Interpretation
The acidic metabolic byproducts produced by species able to ferment the single carbohydrate in
the broth turn the phenol red in the medium yellow. Species unable to ferment the carbohydrate
will not produce acidic byproducts and therefore will leave the medium red to pink in colour.1

1. Food and Drug Administration. (1998). Bacteriological Analytical Manual.

2. National Center for Biotechnology Information. PubChem Compound Database; CID=4766,
https://pubchem.ncbi.nlm.nih.gov/compound/4766 (accessed 14 Mar. 2019).

5.9: Catalase Test

The catalase test is used to determine whether a bacterial species is able to produce the heme
enzyme catalase. Catalase catalyses the decomposition of hydrogen peroxide, a reactive
oxygen species, into water and oxygen gas.2 Reactive oxygen species like H2O2 cause oxidative
damage through free radical generation, which generally leads to cell death.1 Below is the
chemical reaction catalysed by catalase.

2 H2O2 --(catalase)--> 2 H2O + O2

Use

There are two methods in which the catalase test may be performed, depending on the nature
of your source culture.

From an agar culture: A small drop of hydrogen peroxide is placed on a clean microscope slide.
Using a plastic disposable loop, pick up some of the bacterial growth from the agar and smear
the culture into the hydrogen peroxide.

From a broth culture: Collect a small sample in one end of a glass capillary tube, without
blocking this end. Dip the opposite end into hydrogen peroxide, then turn the tube upside-down
(so that the end with the broth culture is pointing downwards). Tap the tube against the lab
bench until the hydrogen peroxide touches the bacterial culture.3
Interpretation

If bubbles form when hydrogen peroxide comes into contact with the bacterial culture, this
denotes a positive result, as the bubbles (effervescence) are evidence of degradation producing
oxygen gas.

1. Murphy, E. & Friedman, A. J. (2019). Hydrogen peroxide and cutaneous biology: Translational applications, benefits, and
risks. Journal of the American Academy of Dermatology. (accepted) doi.org/10.1016/j.jaad.2019.05.030
2. Gebicka, L. & Krych-Madej, J. (2019). The role of catalases in the prevention/promotion of oxidative stress. Journal of
Inorganic Biochemistry 197. (accepted) doi.org/10.1016/j.jinorgbio.2019.110699
3. Fung, D. Y. C. (1994). Rapid methods and automation for seafood microbiology. In A. M. Martin (ed.) Fisheries
Processing: Biotechnological Applications. London, United Kingdom: Chapman & Hall.

5.10: Gelatine Liquefaction Test

The hydrolysis, or liquefaction, of gelatine is tested by adding bacterial culture to a tube of

nutrient gelatine medium. If a bacterial species is able to produce enzymes called gelatinases,
which break gelatine down into individual amino acids, turning the solid structure of gelatine to
liquid.1

Ingredients

Peptone, 5g
Beef extract, 3g
Gelatine, 120g: differential ingredient

Use

Use a sterile inoculating needle to stab one nutrient gelatine tube with bacterial culture. Incubate
this tube and an uninoculated tube (as a control) at 25oC for 48 hours. Check the tubes 24 hours
into the incubation period.

Interpretation

Upon taking the tubes out of the incubator, place them in an ice bath for 15 minutes. Then, tilt
the tubes to see if the gelatine has been hydrolysed-- the control tube should remain solid, and
if the other tube appears liquid, hydrolysis has occurred.2 If no change is seen, continue
checking the tubes up to seven days in case hydrolysis is slow.

1. Greene, RA & Larks, GG. (1955). A quick method for the detection of gelatin liquefying bacteria. Journal of Bacteriology,
77(iss), 60-64.
2. de la Cruz, TEE & Torres, JMO. (2012). Gelatin hydrolysis test protocol. American Society for Microbiology.

5.11: Methyl Red Test

The methyl red test is used to differentiate between members of the family Enterobacteriaceae
based on their fermentation of different sugars. Certain sugars are fermented via the mixed
acids pathway, which yields a large amount of acidic byproducts. These byproducts reduce the
pH of the medium from 6 to 4.4 or lower. When methyl red is added to the medium following
growth, a red colour is produced due to the low pH.

Ingredients

Buffered peptone, 7g
Dipotassium phosphate, 5g
Dextrose, 5g

Use

To inoculate the MRVP broth, tilt the tube and place a loopful of culture against the side of the
test tube, below the level of the liquid. Vortex the tube to ensure the culture is mixed into the
liquid and inoculate at 37oC for 48 hours.

Interpretation

Upon removing the culture from the incubator, add five drops of methyl red reagent from the
refrigerator. If the colour changes from light yellow to red, the dextrose in the medium has been
fermented via the mixed acids pathway (a positive result). If no colour change is seen, this is a
negative result.

1. Clark, WM & Lubs, HA. (1915). The differentiation of bacteria of the colon-aerogenes family by the use of indicators.
Journal of Infectious Disease, 17(iss), 169-173.
2. McDevitt, S. (2009). Methyl red and Voges-Proskauer test protocols. American Society for Microbiology.

5.12: Gelatine Liquefaction Test

The hydrolysis, or liquefaction, of gelatine is tested by adding bacterial culture to a tube of

nutrient gelatine medium. If a bacterial species is able to produce enzymes called gelatinases,
which break down gelatine into individual amino acids, turning the solid structure of gelatine to
liquid.

Ingredients
● Peptone, 5g
● Beef extract, 3g
● Gelatine, 120g: differential ingredient

Use

Use a sterile inoculating needle to stab one nutrient gelatine tube with bacterial culture. Incubate
this tube and an uninoculated tube (as a control) at 25oC for 48 hours. Check the tubes 24 hours
into the incubation period.
Interpretation

Upon taking the tubes out of the incubator, place them in an ice bath for 15 minutes. Then, tilt
the tubes to see if the gelatine has been hydrolysed--the control tube should remain solid, and if
the other tube appears liquid, hydrolysis has occurred. If no change is seen, continue checking
the tubes up to seven days in case hydrolysis is slow.

5.13: Methyl Red Test

The methyl red test is used to differentiate between members of the family Enterobacteriaceae
based on their fermentation of different sugars. Certain sugars are fermented via the mixed
acids pathway, which yields a large amount of acidic byproducts. These byproducts reduce the
pH of the medium from 6 to 4.4 or lower. When methyl red is added to the medium following
growth, a red colour is produced due to the low pH.

Ingredients
● Buffered peptone, 7g
● Dipotassium phosphate, 5g
● Dextrose, 5g

Use

To inoculate the MRVP broth, tilt the tube and place a loopful of culture against the side of the
test tube, below the level of the liquid. Vortex the tube to ensure the culture is mixed into the
liquid and inoculate at 37oC for 48 hours.

Interpretations

Upon removing the culture from the incubator, add five drops of methyl red reagent from the
refrigerator. If the colour changes from light yellow to red, the dextrose in the medium has been
fermented via the mixed acids pathway (a positive result). If no colour change is seen, this is a
negative result.

5.14: Coagulase Test

The coagulase test is used to ascertain whether a culture produces the enzyme coagulase,
which converts fibrinogen to fibrin. Typically, this test is used to differentiate Staphylococcus
aureus, which produces this enzyme, from coagulase-negative Staphylococcus species (though
S. aureus is not the only coagulase positive Staphylococcus species). S. aureus is able to
produce bound coagulase (which is bound to the cell wall, causing fibrinogen to precipitate
against the surface of the cell) and free coagulase (which activates plasma coagulase-reacting
factor (CRP) and complexes with it in order to make a fibrin clot). Bound coagulase may be
detected using a slide coagulase test, while free coagulase may be detected using a tube
coagulase test.
Ingredients
● Human or rabbit plasma (differential ingredient)
● Physiological saline

Use

To conduct the slide test, place a drop of saline on a clean microscope slide. Using a sterile
inoculating loop, carefully pick up some of your test bacteria and mix it into the saline on the
slide. Sterilise the loop. Add a drop of plasma to the suspension and mix it together. Within ten
seconds, you should be able to see clumping if coagulase is present.

To conduct the tube test, dilute the plasma with saline solution and add 500 μL of the diluted
plasma to a clean test tube. To that, add 100 μL each of your test bacteria and sterile TSB. Swirl
to mix. Incubate at 37oC and check the tube after an hour for clotting.

5.15: Procedure

In this lab, you will have a brief introduction to many of these media. Inoculate the following
media:
1. Bile aesculin agar: Inoculate the surface of the agar slant with a loopful of
Enterococcus faecalis. Incubate at 37oC for 48 hours. You should expect to see a
complete blackening of the medium.
2. Blood agar: Using a permanent marker, divide the plate into halves. Label one half ‘SA
- BETA’ and the other ‘SM - ALPHA’. On the ‘SA’ half, use an inoculating loop to streak
Staphylococcus aureus onto the agar surface. On the ‘SM’ half, do the same with
Streptococcus mitis culture. Incubate at 37oC for 24 hours. You should expect to see an
alpha haemolytic pattern around the S. mitis streak and a beta haemolytic pattern
around the S. aureus streak.
3. Eosin methylene blue agar: Using a permanent marker, divide the plate into thirds.
Label one third ‘EC - LAC ++’, one third ‘KA - LAC +’, and the last third ‘ST - LAC -’.
Inoculate the ‘EC’ segment with a loopful of Escherichia coli broth, the ‘KA’ segment with
Klebsiella aerogenes, and the ‘ST’ segment with Salmonella typhi. Incubate at 37oC for
24 hours. You should expect to see iridescent green, dark E. coli growth, pink to purple
K. aerogenes growth, and colourless S. typhi growth.
4. Methyl red test: Use a micropipette to inoculate the methyl red broth with E. coli culture.
Incubate at 37oC for 48 hours. You must add 5 drops of methyl red reagent in order to
see your result-- the broth should turn bright red.
5. Mannitol salt agar: Using a permanent marker, divide the plate into halves. Label one
half ‘SA - MAN +’ and the other ‘SE - MAN -’. Inoculate the ‘SA’ half with a loopful of S.
aureus and the ‘SE’ half with Staphylococcus epidermidis. Incubate at 37oC for 24 hours.
You should expect to see a bright yellow colour change in the agar around the S. aureus
growth, but no colour change around S. epidermidis.
 6. Phenol red fermentation broth: Use a micropipette to inoculate the PRFB broth with E.
coli culture. Incubate at 37oC for 24 hours. You should expect to see a colour change
from pink to yellow, as well as gas bubbles trapped in the Durham tube.
7. Simmons’ citrate agar: Using an inoculation loop, inoculate the slant surface with K.
aerogenes. Incubate at 37oC for 48 hours. You should expect to see a colour change
from green to blue.
8. SIM medium: Use a micropipette to inoculate the broth with E. coli culture. Incubate at
37oC for 48 hours. You must add 1-3 drops of Kovac’s reagent in order to see your
result-- the added reagent should turn bright red.

Assignment

Next week, your Unknown Project will begin. This will entail writing a detailed scientific paper
about the process you could use to determine the species of an unknown sample. This week,
you will use this lab procedure to write a practise Materials & Methods section. Use the following
rubric to format your assignment:

Materials and methods (10 points)

● Materials used; names of reagents and media required (2.25 points)

● Description of each test performed, in the order they were performed (2.25 points)
● Possible outcomes for each test and what each outcome indicates (2.25 points)
● Interpretation of reaction and identification of enzyme/end products (2.25 points)
● Citation(s) of relevant peer-reviewed article(s)/other published works (1 points)

You must use all the tests performed in your lab section to write this assignment-- not just the
one you and your partner performed.