This document provides descriptions of various microbiological tests and media, including what each tests for and how to interpret the results. It explains that Phenol Red Broth tests for carbohydrate fermentation by detecting acid production which turns the broth yellow, Durham tubes test for gas production by detecting bubbles, and O.F. Basal Medium distinguishes between oxidation and fermentation by adding oil to cut off oxygen in one tube. Similarly, it outlines several other common differential and selective tests and media, such as Citrate Agar, Decarboxylase Broth, TSB for indole production, Nitrate Broth, Urease Broth, Blood Agar for hemolysis, PEA agar for gram-

1. Phenol Red Broth . Differential . Tests for - Carbohydrate Fermentation - Gas Production (Durham Tube) . If a microbe ferments carbohydrates, acid gets left behind , this acid turns a Phenol red broth into a yellow color. So if the tube is yellow, carbohydrate fermentation took place (positive reaction). If it is still red, there’s no acid, and no carbohydrate fermentation took place (negative reaction). + - Durham Tube – Ok this ones easy, if you see a bubble at the top of the tube, gas was produced (positive). If you don’t see a bubble, then no gas was produced (negative). + -

2. O.F Basal Medium . Differential .Tests for - Oxidation - Fermentation . To break down sugar with oxidation, microbes utilize…. You guessed it, oxygen. In fermentation, oxygen is not utilized. By adding oil into one inoculated O.F basal tube, which cuts off it’s air supply, and leaving one exposed to oxygen, you can weed out which microbes use oxidation and which ones use fermentation. The way this works is: when bacteria metabolize the sugar in the tube, they leave acid as a byproduct. This acid changes the solution to a yellow color. So results could be: . No Reaction = No color change . Oxidation = Tube without oil turns yellow (usually at surface), oxygen was used to break down the sugar . Fermentation = Both tubes turn yellow. This happens because fermentation produces more acid than oxidation, and any growth below the surface in the tube without oil is Anaerobic.

3. Citrate Agar . Differential . Tests for: Citrus Utilization . This one’s really simple. The citrate slant starts off green. If the slant turns blue, citrate has been utilized (positive). If it stays green, citrate hasn’t been utilized (negative). That’s pretty much it. And now for an advanced illustration: + -

4. Decarboxylase Broth . Differential . Tests for: Carboxylic Acid Removal . In this test, we are looking for bacteria with the enzyme Decarboxylase. This enzyme removes the carboxyl group from the amino acid placed in the tube along with the bacteria. If this enzyme is present in the bacteria, and it does it’s job, Carboxylic acid will be removed. Blah blah blah..what do you need to know? If the tube changes colors, there’s no longer carboxylic acid in it. If there’s no acid In the solution, it’s alkaline, indicated by a shade of purple (positive). If the tube is yellow, the acid is still present, meaning decarboxylase was not present, and no acid was removed (negative). Note: Glucose is in these tubes as well, which is utilized by any bacteria placed in the tube, leaving acid, which turns the solution yellow. This yellow will eventually return It’s original color (in your tubes with POSITIVE results) Note 2: This enzyme works anaerobically (as in - without oxygen) which explains why oil is placed in the tube. + -

5. TSB (Indole) . Differential . Tests for: Indole Production (Tryptophan Catabolization) .Tryptophan is is an amino acid, which can be catabolized (broken down) by certain bacteria. With this test, we are looking for those bacteria. When tryptophan is broken down, indole is released as a by product. To determine whether or not tryptophan was broken down, and indole was released, add Kovac’s or James’ reagant to the solution. These reagants react with indole to form a red ring . So, if kovac’s or james’ reagant is added to TSB, and a red ring forms, indole is present. (postive). If a red ring does not form after adding the reagants, indole is not present, and tryptophan was not catabolized (negative). So:In other words, on the test, TSB with a red ring = Positive. Don’t confuse this with the Nitrate broth (next slide) it'll look somethin like this

6. Nitrate Broth . Differential . Tests For: Nitrate reduction . With the nitrate broth, the goal is to find bacteria that reduce nitrate. Nitrate, when reduced, becomes nitrite, or nitrogen gas. So, to determine whether a microbe reduces nitrate, you must test for the presence of this nitrite, or nitrogen gas. . To do this, Solution A and Solution B are added to the nitrate broth. These solutions will react with nitrite, forming a red ring in the tube. So, if a red ring forms, the specimen is Positive for nitrate reduction. . If a red ring does not develop, it does not mean nitrate was not reduced, although this is a possibility. Zinc is added to a tube with no ring to reduce any nitrate Remaining. If a red ring occurs after the zinc, the specimen is negative for nitrate reduction. If a ring still does not occur after zinc, this means that the nitrate in The tube was reduced to nitrogen gas After Sol. A & B: = Positive After Zinc: = Negative

7. Urease Broth . Differential . Tests for: Urease Production . With the urease broth, the goal is to test bacteria for urease, an enzyme which breaks down urea into ammonia and carbon dioxide. When ammonia and carbon Dioxide are present in a urease broth, the soultion changes to a fuschia pink color, Indicating the presence of urease (positive). If no Urease was in the broth, than Ammonia and carbon dioxide aren’t either, leaving the solution slightly Acidic, with a salmon color (negative). + - bada boom

8. Blood Agar . Differential . Tests For: Presence and degree of Hemolysis . Hemolysis is the catabolization (breaking down) of red blood cells. With the blood agar plate, the goal is not only to identify which bacteria break down red blood cells, but also to what extent. . Bacteria that partially destroy red blood cells are Alpha Hemolytic . This will show up as a green halo adjacent to the bacterial growth. . Bacteria that completely destroy red blood cells are Beta Hemolytic. This will show up as a clear area adjacent to the growth. .Bacteria that don’t break down red blood cells are Non-Hemolytic. The adjacent area Remains red. .Green = Alpha .Clear = Beta .Red = Gamma ; Non-Hemolytic (Shown in front of light source)

9. PEA Agar (Phenylethyl Alcohol) . Selective . Tests for: Gram positive microbial growth . Gram negatives cannot grow on PEA (it dissolves their lipopolysaccharides..which, I guess are important). Anyways, all you need to know is, if something is growing on PEA, it’s gram positive. End of story. Not Gram + Gram +

10. MSA (Mannitol Salt Agar) . Highly Selective / Differential . Tests for: Staphylococci . If something is growing on this medium, it is staphylococci. It’s really salty and Staph is the only bacteria man enough to grow on it. It’s also got mannitol in it, which Is a sugar. What happens when sugar is fermented? Acid, and in the presence of acid the agar changes color. It just so happens, out of the staphylococci, Staph aureus is the only one which ferments mannitol. So if the color changes, you know It’s staph aureus at work. If not, it’s one of the less dangerous staph species. .Growth = Staphylococci .Growth + Color Change = Staph. Aureus .No Growth = Not Staph

11. MAC Agar . Selective / Differential .Tests for: Gram Negative Growth / Lactose Fermentation . This medium inhibits gram positive growth, so if you’ve got growth on it, it’s gram negative bacteria. Also, it’s got lactose in it, so if the bacteria decide to ferment that lactose, the agar will change color, because of the acid which is left behind. So: .Growth = Gram Negative, Lactose Negative .Growth/Color change = Gram Negative / Lactose + .No Growth = Not Gram Negative

12. EMB Agar .Selective / Differential .Tests for: Gram Negative / Lactose Fermentation . EMB is a lot like MAC. They are both selective for gram negatives, and test for lactose Fermentation. The difference though, is that EMB allows some gram positives to grow, And also shows E. Coli and Klebsiella Pneumoniae as metallic green colonies. Growth = Lactose Negative Growth & Color Change = Gram Negative / Lactose Positive Green metallic growth = E. Coli or K. Pneumoniae

13. TSIA . Multitest . Tests For: -Glucose Fermentation / Glucose & Lactose Fermentation -Iron Utilization (hydrogen sulfide production) -Gas production . This one can seem like a lot, but it’s actually pretty simple. We’ll start with the name. TSIA = Triple Sugar Iron.Agar. The three sugars that make up this agar are glucose, lactose and sucrose. You can just forget about sucrose, it doesn’t matter. But glucose and lactose are important. Think of glucose as being the bottom, and lactose as being The slant. So, if the bottom changes colors, it’s glucose positive. If both the slant and Bottom change colors, it’s glucose and lactose positive. As for the iron part of the name..it’s just iron. If iron is utilized by the bacteria, then the whole tube turns black, because the byproduct of iron utilization is hydrogen sulfide. So if the tubes black, it’s got Hydrogen sulfide in it (H2S Positive). Last, if you see any cracks or bubbles, gas was Produced (Co2 +). . Yellow bottom = Glucose + . Yellow slant & bottom = Glucose & Lactose + . Black Medium = H2S + . Cracks or bubbles = Co2 +

15. SIM Agar . Multitest . Tests for: S = Sulfide Production I = Indole Production M = motility . Just like TSIA, this tests for Hydrogen sulfide production, which turns the tube black. It also tests for Indole. If you remember from the TSB, the way to test for indole, Is to add Kovac’s or James’ Reagant to the tube, and see if a red ring develops. A Red ring indicates indole positive. Lastly, if any bacteria is growing away from the Inoculation line, it’s motile. . Black Tube = H2S + . Red ring after reagant = Indole positive . Growth away from line = Motility positive

16. Random Stuff We’ll Probably Have To Know . Differential Medium– allows all to grow, and allows you to differentiate. Ex: + or – . Selective Medium- Allows only a certain type of bacteria. Ex: PEA allows only gram positive . Highly Selective- only grows a very specific bacteria. Ex: Staph. In MSA . Multitest- tests a bunch of stuff. SIM and TSIA are the only two talked about in class . UV light = Bad for you, especially your eyes, and it can rock bacteria’s world, if it’s Uncovered in the petri dish . Obligate (strict) Aerobe- needs oxygen, will always grown on top of medium where there is air. Obviously . Obligate (strict) Anaerobe- needs to stay away from oxygen, or it dies, grows under the surface of a medium where there is no air.

17. Random Stuff We’ll Probably Have To Know Pt. 2 .Microaerophile- needs oxygen, but not much . Aerotolerant- Doesn’t care whether oxygen lives or dies. Doesn’t use it and isn’t harmed by it . Facultative Anaerobe- Anaerobe that can use oxygen if it feels like it, but doesn’t Have to. .Capneic- need higher than atmospheric levels of Co2 . Spores- are pretty much resistant to everything . Zone of inhibition- area next to antibiotic/disinfectant where nothing grows . If little bacteria colonies are forming in the zone of inhibition, that strain is becoming resistant. . Resistant- antibiotic agent is ineffective, little or no zone of inhibition

18. Random Stuff We’ll Probably Have To Know Pt. 3 .Sensitive- large zone of inhibition, the bacteria get dominated by the antibiotic agent . Susceptible- smaller zone of inhibition . Washing your hands makes more bacteria grow on it because your hands are wet. . Fomites are inanimate objects that can carry microbes and don’t feed them. Nonliving and nonedible. Bacteria cannot grow on fomites. . Reservoirs of infection are places bacteria can grow with nutrition, humans, animals, Food, etc etc etc.. . Read all of the chapters (especially the bold stuff) and questions from the lab Manuals, and try to remember all the stuff that was emphasized in class. I guess.. I really don’t know, I have no clue, just guessing here.