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BIO203 Laboratory Media and Biochemical Tests

Table of Contents I. Media 1

TSA – Tryptic Soy Agar 1

Blood Agar 2

EMB – Eosin Methylene Blue Agar 3

MSA – Mannitol Salt Agar 4

MacConkey Agar 5

II. Colony Morphology of 12 bacteria in our laboratory 6

III. Biochemical Tests 15

Motility Test 15

Oxidase Test 16

Glucose Fermentation Test 17

Nitrate Test 18

FT – Fluid Thioglycollate Test 19

Urea Test 20

IMViC: Tryptone broth/Indole test (I) 21

IMViC: MR-Methyl Red (“M”) 22

IMViC: VP-Voges-Proskauer Test (V) 23

IMViC: Simmons citrate slant (C) 24

IMViC test results for specific bacteria 25

1

MEDIA

Isolation of bacteria is accomplished by growing ("culturing") them on the surface of

solid nutrient media. Such a medium normally consists of a mixture of protein

digests (e.g., peptone, tryptone) and inorganic salts, hardened by the addition of

1.5% agar. Standard or general purpose media will support the growth of a wide

variety of bacteria – for example: TSA. A medium may be enriched by the addition

of blood or serum – we use sheep’s blood agar.

Selective media contain ingredients that inhibit the growth of some organisms but

allow others to grow. For example, mannitol salt agar contains a high concentration

of sodium chloride that inhibits the growth of most organisms but permits

staphylococci to grow.

Differential media contain compounds that allow groups of microorganisms to be

visually distinguished by the appearance of the colony or the surrounding media,

usually on the basis of some biochemical difference between the two groups. Blood

agar is one type of differential medium, allowing bacteria to be distinguished by the

type of hemolysis produced. Some differential media are also selective, for example,

eosin-methylene blue agar, which is selective for gram-negative coliforms and can differentiate lactose-fermenting and non-lactose-fermenting bacteria.

Examples of the agars and some of the bacteria that you will see in this class:

TSA – Tryptic Soy Agar: used for the isolation and cultivation of nonfastidious and fastidious

microorganisms – not the medium of choice for anaerobes.

TSA Streak Plate: pigmented Serratia

marcescens

TSA Streak Plate: Stapholococcus

aureus

2

Blood Agar – used for the detection of hemolytic activity of microorganisms.

alpha (α) hemolysis: green halo around colony.

beta (β) hemolysis: a clear, colorless zone appears around the colonies, indicating the red blood cells have undergone complete lysis.

gamma (γ) hemolysis: normal-looking colony.

Blood agar – Staphylococcus aureus

Blood agar Micrococcus luteus

Blood agar – Streptococcus

pneumoniae

3

EMB – Eosin Methylene Blue Agar: selective and differential medium. Eosin differentiates

between two major coliforms: E. coli (smaller, green-metallic sheen) and Enterobacter

aerogenes (larger, rose color). Methylene blue selectively inhibits the growth of Gram+

bacteria. With this media we can also determine which bacteria are Gram-negative and which

are Gram-positive, because only Gram-negative bacteria grow on this special media. The

enhanced cell walls of Gram-negative bacteria protect these bacteria from the dye in the EMB

plates. The dye is able to enter the cells of Gram positive bacteria and kill them.

EMB: Escherichia coli

EMB: A=Escherichia coli

B=Pseudomonas

C=Klebsiella

D=Enterobacter aerogenes

4

MSA – Mannitol Salt Agar: Demonstrates the ability of a bacterium to grow in a 7.5%

salt environment (growth indicates tolerance for high salt environment – no growth

shows intolerance/no determination of ability to ferment mannitol w/ no growth).

Demonstrates the ability of a bacterium to ferment mannitol (“+” mannitol

fermentation = yellow due to lowered pH from acids in waste. “-“ mannitol

fermentation = no color change/no acid).

MSA – Staphylococcus epidermidis

MSA – Staphylococcus aureus

5

MacConkey Agar: Demonstrates the ability of a gram negative bacterium to

metabolize Lactose. MacConkey agar is both a selective and differential medium

frequently used in culture testing. It contains crystal violet dye and bile salts, both

of which inhibit the growth of most gram-positive bacteria. It contains lactose (a

sugar) and neutral red indicator (a pH indicator which is yellow in a neutral solution,

but turns pink to red in an acidic environment), which allow for differentiation. On

MacConkey agar, Escherichia coli and Enterobacter aerogenes would ferment the

lactose producing acid and would form colonies pink to red in color. On the same

medium, Salmonella, Shigella, and Pseudomonas species would not ferment the

lactose and would form off-white colonies. The red colored colonies show that acid

was produced from lactose, meaning the bacteria could utilize lactose as a carbon

source.

Quadrant 1: Growth on the plate indicates the organism, Enterobacter aerogenes,

is not inhibited by bile salts and crystal violet and is a gram-negative bacterium.

The pink color of the bacterial growth indicates E. aerogenes is able to ferment lactose.

Quadrant 2: Growth on the plate indicates the organism, Escherichia coli, is not

inhibited by bile salts and crystal violet and is a gram-negative bacterium. The pink color of the bacterial growth indicates E. coli is able to ferment lactose.

Quadrant 3: Absence of growth indicates the organism, Staphylococcus epidermidis,

is inhibited by bile salts and crystal violet and is a gram-positive bacterium.

Quadrant 4: Growth on the plate indicates the organism, Salmonella typhimurium,

is not inhibited by bile salts and crystal violet and is a gram-negative bacterium.

The absence of color in the bacterial growth indicates S. typhimurium is unable to ferment lactose.

6

COLONY MORPHOLOGY

Bacillus subtilis

Colonies which are dry, flat, and irregular, with lobate margins.

Proteus vulgaris

7

Pseudomonas

This gram negative rod forms mucoid colonies with umbonate elevation.

Staphylococcus aureus

Circular, pinhead colonies which are convex with entire margins. This gram positive coccus often

produces colonies which have a golden-brown color.

8

Mycobacterium smegmatis. Note the waxy appearance of this Acid-fast

bacterium.

9

Escherichia coli

This gram negative rod (coccobacillus) forms shiny, mucoid colonies which have entire margins

and are slightly raised. Older colonies often have a darker center.

10

Serratia marcescens. These gram negative rods produce mucoid colonies which have entire

margins and umbonate elevation. Note that there are both red and white colonies present on

this plate. Some strains of S. marcescens produce the red pigment prodigiosin in response to

incubation at 30o C, but do not do so at 37

o C. This is an example of temperature-regulated

phenotypic expression.

11

Corynebacterium xerosis

Lactobacillus

12

Staphylococcus epidermidis

Circular, pinhead colonies which are convex with entire margins. The colonies of this gram-

positive coccus appear either the color of the agar, or whitish.

13

Micrococcus luteus. Circular, pinhead colonies which are convex with entire margins. This gram

positive coccus produces a bright yellow, non-diffusable pigment.

14

Enterobacter aerogenes. This gram negative rod is a common contaminant of vegetable matter

which forms shiny colonies with entire margins and convex elevation.

15

Biochemical Tests

Motility Agar: identifies the ability bacteria to move (i.e. flagellated cells). We use plates

instead of tubes.

Non-motile – no

movement from

stab

Motile – growth

far away from

stab

16

Oxidase Test: Used to demonstrate the ability of a bacterium to produce the enzyme

cytochrome- c oxidase, capable of reducing oxygen. Only Aerobic bacteria have this enzyme.

This test will distinguish Aerobic vs. Anaerobic metabolism. A positive test will show a color

change to blue, then to dark purple or black, within 10 to 30 seconds.

17

Glucose Fermentation tubes: determines the ability of a bacterium to ferment the sugar glucose

as well as its ability to convert end products (pyruvic acid) into gaseous byproducts. Phenol red

indicator is used to show acid fermentation (yellow below pH 6.8) or alkaline fermentation (red

above pH 8.4). Durham tubes collect CO2 gas produced from fermentation process.

Phenol Red + Sugar:

Left = nonfermenter of glucose, no

gas produced.

Middle = glucose fermentation, but

no gas produced.

Right = glucose fermentation and gas

production.

18

N -Nitrate broth: demonstrates the ability of a bacterium to produce the enzyme nitratase,

capable of converting nitrate to nitrite.

1. Red color after addition of Nitrate A + Nitrate B = positive

test for nitrate reduction (end)

2. No color change after addition of Nitrate A + Nitrate B =

possibly negative for nitrate reduction (continue to next

step to confirm)

a) Red color after adding Zn = confirmed negative

for nitrate reduction

b) No color change after adding Zn = positive test

for nitrate reduction (organism reduced nitrate

to nitrite then further to ammonia or N2). May

see gas bubble in Durham tube.

19

FT – Fluid Thioglycollate tubes: show oxygen usage of bacteria by where they grow in the tube.

Resazurin is an indicator that is pink in the presence of oxygen (notice upper portion of tube

where media touches air – if it is pink deeper in the tube, the tube has too much oxygen

diffused and should not be used). Thioglycollate binds w/ oxygen that diffuses into the media,

making it unavailable deeper in the tube.

Tube 1 = obligate anaerobe. Growth in the bottom but none in the top.

Tube 2 and 5(mislabeled!!) = obligate aerobe. Growth only at the top.

Tube 3 = aerotolerant facultative anaerobe. Dense growth throughout tube.

Tube 4 = facultative anaerobe. Growth throughout, but more dense at top.

20

U -Urea broth: demonstrates the ability of a bacterium to produce the enzyme urease, capable

of hydrolyzing urea. Phenol red indicator is added (fuchsia above pH 8.4) to show rise in pH due

to accumulation of ammonia.

21

IMViC: A battery of biochemical tests known as IMViC are used in the clinical lab to distinguish

between enteric microorganisms. The acronym IMViC stands for indole, methyl red, Voges-

Proskauer and citrate. The "i" in the acronym is added for pronunciation purposes.

Tryptone broth/Indole test (“I”): Used to demonstrate the ability of a bacterium to produce the

enzyme tryptophanase. This enzyme acts on the amino acid to produce “indole”.

Tryptone

broth/Indole –

positive result

Tryptone

broth/Indole –

negative result

22

Methyl Red (“M”) – an indicator of low pH (red below pH of 4.4) – used to show the mixed acid

fermentation ability of bacteria.

23

VP -Voges-Proskauer Test (“Vi”) – used to show bacterial production of acetoin, also known as

2,3-butanediol.

24

Simmons citrate slant (“C”) – Simmons citrate agar tests for the ability of a gram-negative

organism to import citrate for use as the sole carbon and energy source. Only bacteria that can

utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate

medium, thus a citrate-negative test culture will be virtually indistinguishable from an

uninoculated slant.

Simmons citrate – blue is a positive

citrate test, while green is

negative/no growth

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IMViC test results for specific bacteria:

Escherichia coli: Tube A: + Idole Tube B: + methyl red Tube C: - VP Tube D: - citrate

Proteus vulgaris: Tube A: + Idole Tube B: + methyl red Tube C: - VP Tube D: - citrate

Citrobacter freundii: Tube A: - Idole Tube B: + methyl red Tube C: - VP Tube D: + citrate

Enterobacter aerogenes: Tube A: - Idole Tube B: - methyl red Tube C: + VP Tube D: + citrate