BIO203 Laboratory Media and Biochemical Tests

Table of Contents
I. Media
TSA – Tryptic Soy Agar Blood Agar EMB – Eosin Methylene Blue Agar MSA – Mannitol Salt Agar MacConkey Agar

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II. Colony Morphology of 12 bacteria in our laboratory III. Biochemical Tests
Motility Test Oxidase Test Glucose Fermentation Test Nitrate Test FT – Fluid Thioglycollate Test Urea Test IMViC: Tryptone broth/Indole test (I) IMViC: MR-Methyl Red (“M”) IMViC: VP-Voges-Proskauer Test (V) IMViC: Simmons citrate slant (C) IMViC test results for specific bacteria

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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

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indicating the red blood cells have undergone complete lysis. alpha (α) hemolysis: green halo around colony. colorless zone appears around the colonies. Blood agar Micrococcus luteus 2 . Blood agar – Staphylococcus aureus gamma (γ) hemolysis: normal-looking colony.Blood Agar – used for the detection of hemolytic activity of microorganisms. Blood agar – Streptococcus pneumoniae beta (β) hemolysis: a clear.

coli (smaller. EMB: Escherichia coli EMB: A=Escherichia coli B=Pseudomonas C=Klebsiella D=Enterobacter aerogenes 3 .EMB – Eosin Methylene Blue Agar: selective and differential medium. rose color). 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. green-metallic sheen) and Enterobacter aerogenes (larger. Methylene blue selectively inhibits the growth of Gram+ bacteria. Eosin differentiates between two major coliforms: E. The dye is able to enter the cells of Gram positive bacteria and kill them. With this media we can also determine which bacteria are Gram-negative and which are Gram-positive.

“-“ mannitol fermentation = no color change/no acid).5% salt environment (growth indicates tolerance for high salt environment – no growth shows intolerance/no determination of ability to ferment mannitol w/ no growth). MSA – Staphylococcus epidermidis MSA – Staphylococcus aureus 4 . Demonstrates the ability of a bacterium to ferment mannitol (“+” mannitol fermentation = yellow due to lowered pH from acids in waste.MSA – Mannitol Salt Agar: Demonstrates the ability of a bacterium to grow in a 7.

On the same medium. which allow for differentiation. It contains crystal violet dye and bile salts. and Pseudomonas species would not ferment the lactose and would form off-white colonies. It contains lactose (a sugar) and neutral red indicator (a pH indicator which is yellow in a neutral solution. Salmonella typhimurium. is not inhibited by bile salts and crystal violet and is a gram-negative bacterium. Quadrant 4: Growth on the plate indicates the organism. both of which inhibit the growth of most gram-positive bacteria. 5 . is not inhibited by bile salts and crystal violet and is a gram-negative bacterium. Escherichia coli and Enterobacter aerogenes would ferment the lactose producing acid and would form colonies pink to red in color. coli is able to ferment lactose. Quadrant 3: Absence of growth indicates the organism. Staphylococcus epidermidis. aerogenes is able to ferment lactose. The pink color of the bacterial growth indicates E. but turns pink to red in an acidic environment). Quadrant 1: Growth on the plate indicates the organism. The red colored colonies show that acid was produced from lactose. Enterobacter aerogenes.MacConkey Agar: Demonstrates the ability of a gram negative bacterium to metabolize Lactose. meaning the bacteria could utilize lactose as a carbon source. Shigella. is inhibited by bile salts and crystal violet and is a gram-positive bacterium. On MacConkey agar. MacConkey agar is both a selective and differential medium frequently used in culture testing. Quadrant 2: Growth on the plate indicates the organism. is not inhibited by bile salts and crystal violet and is a gram-negative bacterium. Salmonella. Escherichia coli. The absence of color in the bacterial growth indicates S. typhimurium is unable to ferment lactose. The pink color of the bacterial growth indicates E.

Proteus vulgaris 6 . with lobate margins. flat. and irregular.COLONY MORPHOLOGY Bacillus subtilis Colonies which are dry.

Staphylococcus aureus Circular. 7 .Pseudomonas This gram negative rod forms mucoid colonies with umbonate elevation. This gram positive coccus often produces colonies which have a golden-brown color. pinhead colonies which are convex with entire margins.

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

mucoid colonies which have entire margins and are slightly raised. Older colonies often have a darker center. 9 .Escherichia coli This gram negative rod (coccobacillus) forms shiny.

marcescens produce the red pigment prodigiosin in response to incubation at 30o C. This is an example of temperature-regulated phenotypic expression. Some strains of S. but do not do so at 37o C. These gram negative rods produce mucoid colonies which have entire margins and umbonate elevation. 10 .Serratia marcescens. Note that there are both red and white colonies present on this plate.

Corynebacterium xerosis Lactobacillus 11 .

The colonies of this grampositive coccus appear either the color of the agar. pinhead colonies which are convex with entire margins. 12 . or whitish.Staphylococcus epidermidis Circular.

Circular. 13 . non-diffusable pigment. pinhead colonies which are convex with entire margins.Micrococcus luteus. This gram positive coccus produces a bright yellow.

This gram negative rod is a common contaminant of vegetable matter which forms shiny colonies with entire margins and convex elevation.Enterobacter aerogenes. 14 .

We use plates instead of tubes.e. flagellated cells). Non-motile – no movement from stab Motile – growth far away from stab 15 .Biochemical Tests Motility Agar: identifies the ability bacteria to move (i.

then to dark purple or black. within 10 to 30 seconds. This test will distinguish Aerobic vs. 16 .Oxidase Test: Used to demonstrate the ability of a bacterium to produce the enzyme cytochrome. capable of reducing oxygen.c oxidase. Only Aerobic bacteria have this enzyme. Anaerobic metabolism. A positive test will show a color change to blue.

Durham tubes collect CO2 gas produced from fermentation process.8) or alkaline fermentation (red above pH 8. Phenol red indicator is used to show acid fermentation (yellow below pH 6. 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. but no gas produced. no gas produced. Phenol Red + Sugar: Left = nonfermenter of glucose. Right = glucose fermentation and gas production.4). Middle = glucose fermentation.

Red color after addition of Nitrate A + Nitrate B = positive test for nitrate reduction (end) 2. 18 . May see gas bubble in Durham tube. 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). 1. capable of converting nitrate to nitrite.N -Nitrate broth: demonstrates the ability of a bacterium to produce the enzyme nitratase.

Tube 1 = obligate anaerobe. the tube has too much oxygen diffused and should not be used). Thioglycollate binds w/ oxygen that diffuses into the media. Tube 3 = aerotolerant facultative anaerobe. Tube 2 and 5(mislabeled!!) = obligate aerobe. Growth throughout. 19 . Tube 4 = facultative anaerobe. but more dense at top. making it unavailable deeper in the tube. Growth in the bottom but none in the top. Growth only at the top.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. Dense growth throughout tube.

U -Urea broth: demonstrates the ability of a bacterium to produce the enzyme urease. Phenol red indicator is added (fuchsia above pH 8. 20 . capable of hydrolyzing urea.4) to show rise in pH due to accumulation of ammonia.

Tryptone broth/Indole – positive result Tryptone broth/Indole – negative result 21 . methyl red. VogesProskauer and citrate. The "i" in the acronym is added for pronunciation purposes. The acronym IMViC stands for indole. 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”.IMViC: A battery of biochemical tests known as IMViC are used in the clinical lab to distinguish between enteric microorganisms.

4) – used to show the mixed acid fermentation ability of bacteria.Methyl Red (“M”) – an indicator of low pH (red below pH of 4. 22 .

VP -Voges-Proskauer Test (“Vi”) – used to show bacterial production of acetoin. 23 . also known as 2.3-butanediol.

Only bacteria that can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium. Simmons citrate – blue is a positive citrate test. thus a citrate-negative test culture will be virtually indistinguishable from an uninoculated slant.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. while green is negative/no growth 24 .

VP Tube D: .citrate Proteus vulgaris: Tube A: + Idole Tube B: + methyl red Tube C: .VP Tube D: .citrate Citrobacter freundii: Tube A: .IMViC test results for specific bacteria: Escherichia coli: Tube A: + Idole Tube B: + methyl red Tube C: .methyl red Tube C: + VP Tube D: + citrate 25 .Idole Tube B: + methyl red Tube C: .Idole Tube B: .VP Tube D: + citrate Enterobacter aerogenes: Tube A: .

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