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 0
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 1
Blood Agar used for the detection of hemolytic activity of microorganisms. alpha (α) hemolysis: green halo around colony. Blood agar Streptococcus pneumoniae beta (β) hemolysis: a clear, colorless zone appears around the colonies, indicating the red blood cells have undergone complete lysis. Blood agar Staphylococcus aureus gamma (γ) hemolysis: normal-looking colony. Blood agar Micrococcus luteus 2
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 3
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 4
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. 5
COLONY MORPHOLOGY Bacillus subtilis Colonies which are dry, flat, and irregular, with lobate margins. Proteus vulgaris 6
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. 7
Mycobacterium smegmatis. Note the waxy appearance of this Acid-fast bacterium. 8
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. 9
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 30 o C, but do not do so at 37 o C. This is an example of temperature-regulated phenotypic expression. 10
Corynebacterium xerosis Lactobacillus 11
Staphylococcus epidermidis Circular, pinhead colonies which are convex with entire margins. The colonies of this grampositive coccus appear either the color of the agar, or whitish. 12
Micrococcus luteus. Circular, pinhead colonies which are convex with entire margins. This gram positive coccus produces a bright yellow, non-diffusable pigment. 13
Enterobacter aerogenes. This gram negative rod is a common contaminant of vegetable matter which forms shiny colonies with entire margins and convex elevation. 14
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 15
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. 16
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 CO 2 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. 17
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 N 2 ). May see gas bubble in Durham tube. 18
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. 19
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. 20
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 21
Methyl Red ( M ) an indicator of low ph (red below ph of 4.4) used to show the mixed acid fermentation ability of bacteria. 22
VP -Voges-Proskauer Test ( Vi ) used to show bacterial production of acetoin, also known as 2,3-butanediol. 23
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 24
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 Enterobacter aerogenes: Tube A: - Idole Tube B: - methyl red Tube C: - VP Tube C: + VP Tube D: + citrate Tube D: + citrate 25