Notes for the 3rd week of lab Feb. 12 and 14

Types of Media ( Exercise 7 in Kleyn) -  A Mannitol Salt Agar plate (MSA)  will be used instead of a Glucose Mineral Agar plate. The MSA and the Eosin Methylene Blue plate (EMB) are described  in detail below.  You will be using both of these plates in other exercises during the semester so make sure you understand how they work.

MSA Plate -  This plate contains mannitol, 7.5% sodium chloride, and phenol red indicator.  The NaCl inhibits organisms other than staphylococci.  If the organism growing on the plate can ferment mannitol, the phenol red in the medium changes color from red to yellow.

The first figure below shows what Staphylococcus aureus looks like growing on a MSA plate.

And Staphylococcus epidermidis is growing on the MSA plate below.

EMB Plate - This medium is selective as well as differential.  The medium favors the growth of  gram negative organisms since methylene blue and eosin in the medium inhibits gram positive organisms.  It also contains lactose which lets one distinguish a  lactose fermenter from a nonfermenter.  The colonies of  lactose fermenting organisms can range in appearance from very pink in color to a  purple/blackish color.  Sometimes the organism releases so much acid during lactose fermentation that dyes in the medium begin to precipitate over the colony surface to produce a green metallic sheen.   This is common with E.coli  and can also be seen sometimes with Citrobacter. Examples of lactose fermenters and nonfermenters on EMB plates can be seen in the figures below.

Left Plate - The organism growing on this EMB plate does not ferment lactose since the colonies are white.  Sometimes colonies of a  nonfermenter will appear to have a little pink color due to the color of the medium but do not confuse this with a much darker color due to fermentation.  Try to look at  plates of a fermenter and nonfermenter  side by side to see the difference. 

Right Plate - The organism growing on this EMB plate does ferment lactose.  The colonies have a  purple/black color.

 E.coli which is growing on the EMB plate below shows the green metallic sheen that you sometimes see when cells release a lot of  acid during lactose fermentation. 

 

 
Morphological and Physiological Characteristics of Enteric Microorganisms  - For this portion of lab you will work in pairs.  Each pair will be given one of seven different enteric organisms and will use that organism to inoculate various media as described in your manual. Next week you will interpret the results of your inoculations and as a class you will create a table of test results for all seven organisms. You will use this table as a reference when characterizing your enteric "unknown" which you will be given next week in lab.  You can make a print-out of the following template for recording class test results. 

Your TA will describe how to perform the inoculations and review with you what each test result will tell about the physiology of the organism. The following is a list of the various media that you will be inoculating so that you can review those specific sections of your manual.  

Slants  -1. Kligler's Iron Agar  2. Simmons Citrate  3. Semisolid motility tube

Broths - 1. Glucose  2. Lactose   3. Mannitol  4. MR-VP   5. Sucrose  6. Nutrient  7. Tryptone  8. Urea

Plates - 1. Nutrient  2. Eosin Methylene Blue (EMB)

You will also test for catalase and use the filter paper method to test for oxidase.

Motlity (Exercise 18) - you will test for motility using the tube method described in the manual. If organisms are motile in the semisolid tube they will move out from the stab line while the nonmotile organisms grow within the stab line.  Sometimes it is difficult to see movement clearly so 2,3,5 -Triphenyltetrazolium chloride (TTC) was added to the semisolid medium.  The TTC is used by bacteria as an electron acceptor.  In its oxidized form it is colorless and will become red after it is reduced by the growing cells.  If you see a red stab line only that would mean cells are not moving from the initial stab.   If the tube has a diffuse red color that would mean the cells are moving out from the stab line in all directions.  You can also check the surface of the agar and usually there is bacterial growth all over the surface with motile cells whereas the nonmotile cell will grow on the surface over the stab line only. See the figure below which shows each type of organism.                                                            

It is important that your  stab in  the semisolid agar is in the center of the tube and is as straight as possible.  To do this you might keep your elbows on the table to steady your hands as you stab the agar. You also should make  sure that  your needle is straight. Use the forceps in the drawer to straighten it if needed.   Only stab the medium once with the microorganism.

The organism in the tube on the left in the figure below is motile and the organism in the tube on the right is nonmotile.