Week 5 B Exp 10: Gram Stain Gram Stain: Gram + SE, SA Gram - EC, PA, PV, EA Look at slides and record observation in journal and chart. Exp 14: Differential & Selective Media Media Sheets Handouts Inoculate 5 different media with organisms. Next Week Lab Practical – Gram Stain & SPD techniques Journal check 1st 2nd 10/09

Exp 10: GRAM STAIN TECHNIQUE Media: cultures of SA, SE, EC, PA, PV, EA Procedure: Smear, air dry, heat fix Primary stain: CV (60 sec) - Rinse Mordant: Iodine (60 sec) - Rinse Decolorizing agent: 95% Alcohol (10 sec) - Rinse Counter stain: Safranin (45-60 sec) Gram pos: blue/violet Gram neg: pink/red If they have time to make more slides then required, they may make them. 5. Also, if students want to save slides that they may not have had a chance to view, we have small plastic cases that will hold up to 5 slides. 6. The students can put their name and section on a piece of tape and stick it to the case for future viewing. 7.The slide must be at least heat fixed to be saved or stained but no oil on it. 8. The immersion oil is not to be kept in their drawers. We have already had a spill in a drawer. Please put all bottles of immersion oil and also the stains back wear they are kept. Rinse Blot dry - Observe and record 1000x oil immersion

How to Identify or Differentiate an Organism (based on what we have learned to date) Then use reference sources: Bergey’s Manual, Lab Manual & Journal

Differential, Selective, and Enriched Media MacConkey Agar Mannitol Salt Agar General Growth If a medium is selective, that means that it grows only certain types of microbes and inhibits the growth of other types of microbes. A growth medium is considered differential if, when certain microbes grow on that medium, it exhibits a color change that gives us information about the type of microbe growing there. Enriched Blood Agar

Selective Media a. Nutritive Agar b. Phenylethyl Alcohol Agar (PEA) This culture medium allows the growth of G(+) and inhibits the growth of G(-). It is present in this medium at 0.025%, if the concentration was higher it would affect both Gram positive and Gram negative cells. Phenylethyl alcohol interferes with DNA synthesis of G(-) species preventing lipid formation. The plates to the right illustrate PEA inhibition of Gram-negative cell growth.  Plate a is a nutrient agar plate illustrating normal growth of each of the same bacteria. Plate b is a PEA plate with a Gram positive organism on the left and a Gram negative organism on the right.    Notice that the Gram-negative organism shows inhibited growth on the PEA plate a. Nutritive Agar b. Phenylethyl Alcohol Agar (PEA)

Selective/Differential Media Mannitol Salt Agar (MSA) is both selective and differential.  It contains 7.5% sodium chloride which selects for organisms that are halotolerant.  It also contains the carbohydrate mannitol, and a pH indicator, phenol red, which allow distinction between organisms which can ferment mannitol and those that can not.  Organisms that are able to ferment the mannitol will produce acid fermentation products which lower the pH, causing the phenol red indicator to turn yellow. The organic acid metabolites changes the pH indicator in the agar from red to yellow. Pathogenic Staph, such as Staphylococcus aureus are mannitol fermenters, whereas nonpathogenic Staph, like the S. epidermis normal flora that grows on our skin, does not ferment mannitol. On this plate of MSA, S. aureus, a pathogenic member of the genus Stapylococcus is growing on the left side of the plate. On the right side of the plate is its nonpathogenic cousin, S. epidermis, which is not a mannitol fermenter and therefore does not cause the agar to change color. Mannitol Salt Agar (MSA)

Selective/Differential Media MacConkey's Is Selective  MacConkey's is a selective medium because it inhibits the growth of Gram positive bacteria due to the presence of crystal violet and bile salts.  MacConkey's Is Differential  It is also a differential medium, meaning that it differentiates or distinguishes groups of bacteria on the basis of some color reaction on or in the media.  MacConkey’s contains two additives that make it differential:   neutral red (a pH indicator) and lactose (a disaccharide). Bacteria, known as “lactose fermenters”, eat the lactose and in the process create an acidic end product that causes the pH indicator, neutral red, to turn red.  It is the actual colonies of lactose fermenting bacteria that appear pink. Non-lactose fermenting bacteria will be colorless (or, if they have any color, it will be a color other than pink).    Image: MacConkey's Plates:Gram negative, lactose fermenting E. coli in plate on right (pink) and Gram-negative, non-lactose fermenting Salmonella in plate on left (tan/colorless). MacConkey Agar (MAC)

Selective/Differential Media Eosin Methylene Blue (EMB) [Leveine] agar is both selective and differential.  It contains the dyes eosin and methylene blue, which inhibit the growth of gram-positive bacteria and therefore select for gram-negative bacteria.  It also contains the carbohydrate lactose, which allows differentiation of gram-negative bacteria based on their ability to ferment lactose. Quadrant 1:  Growth on the plate indicates the organism, Escherichia coli, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The green metallic sheen indicates E. coli is able to ferment lactose to produce strong acid end-products. Quadrant 2:  Growth on the plate indicates the organism, Pseudomonas aeruginosa, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The absence of color in the bacterial growth indicates P. aeruginosa is unable to ferment lactose. Quadrant 3: Growth on the plate indicates the organism, Enterobacter aerogenes, is not inhibited by eosin and methylene blue and is a gram-negative bacterium.  The pink color of the bacterial growth indicates E. aerogenes is able to ferment lactose to produce weak acid end-products. Quadrant 4:  Absence of growth indicates the organism, Staphylococcus aureus, is inhibited by eosin and methylene blue and is a gram-positive bacterium. Eosin Methylene Blue Agar (EMB)

Enriched Media Blood Agar Blood Agar Is NOT Selective Blood agar is an enriched medium that provides an extra rich nutrient environment for microbes.  Therefore, it is not selective, since it supports the growth of a wide range of organisms.   Blood Agar Is Differential  Blood agar is, however, a differential medium.  Microbiologists use blood agar to distinguish clinically significant bacteria from throat and sputum cultures. Blood agar contains 5% sheep blood. Certain bacteria produce enzymes (hemolysins) that act on the red cells to lyse, or break the blood cells down.  Hemolysis Patterns of Blood Agar Beta hemolysis means that the bacteria's hemolytic enzymes completely beak down the blood cells. The Beta hemolysis pattern results in the media displaying clear halos around bacteria colonies that exhibit B-hemolysis. Alpha hemolysis means that the bacteria can only partially break down the blood cells. This results in the media showing a yellowish/greenish/brownish discoloration (like a bruise) around the colony indicating incomplete hemolysis. Gamma hemolysis means that the bacteria have no effect on the red blood cells and there is no change to the color of the medium.  When Is Blood Agar Used? Blood agar is usually inoculated from a patient’s throat swab, because the medical lab is  trying to detect the presence of Group A beta hemolytic Streptococci (a Gram-positive cocci that causes Beta hemolysis on blood agar.) The major human pathogen in this group is Streptococcus pyogenes, the causative agent of strep throat. Normal throat flora will exhibit alpha or gamma hemolysis. The blood can be from rabbit or sheep. Rabbit blood is preferred if the target bacterium is from the group known as group A Streptococcus. Sheep blood is preferred if the target bacterium is Haemophilus parahaemolyticus. Blood agar is a rich food source for bacteria. So, it can be used for primary culturing, that is, as a means of obtaining as wide a range of bacterial growth from a sample as possible. It is typically not used for this purpose, however, due to the expense of the medium. Other, less expensive agars will do the same thing. What blood agar is uniquely suited for is the determination of hemolysis. Hemolysis is the break down of the membrane of red blood cells by a bacterial protein known as hemolysin, which causes the release of hemoglobin from the red blood cell. Blood Agar

Happy Bacteria E coli Smiley Face on MAC

Exp 14: Differential, Selective and Enriched Media Culture: Gram (+): SE, SA Gram (-): EC, EA, PA, PV Per Table Media: 2 culture each of PEA, MSA, MAC, EMB, Blood Procedure: Lab One pg 102-103 Properly label cover of agar dish. Divide the bottom of the agar dish in 4 quadrants and label with different organism. Streak inoculate each quadrant (single line). Do not stab blood agar. Incubate at 37C for 24 hrs. EC EA control SE PV PA control SA