1. Review of Gram Stain Selective and differential Media
Microbiology
Review of Gram Stain
Selective and differential Media

2. Gram Positive Bacteria
Staphylococcus aureus
Streptococcus salivarius
Bacillus subtilis
Staphylococcus epidermidis

3. Gram Positive Cell Wall

4. Gram Negative Bacteria
E. coli
Pseudomonas aeruginosa
Proteus vulgaris
Enterobacter aerogenes

5. Gram Negative Cell Wall

6. The Smear
1.  Label the frosted side of your slide with your initials, the name of the organism, and the date.  On this side draw a circle in the clear section of the slide.
2.  Turn the slide over.  You will make your smear on this side.
*****If you are using broth follow these directions
Flame your inoculating loop.
Use aseptic technique and remove the top of the culture tube, flame the mouth or the culture tube, and dip the loop into the broth.  Make sure that the loop is filled.
Transfer the loopful of broth and bacteria to the slide. Using a circular motion, spread the broth on the slide.
This is now a " smear"
Allow the smear to dry
When the smear has been allowed to " air dry" , pass the smear through the flame to " heat fix" - Heat fixation causes the proteins and cell parts to coagulate and stick to the slide.
Let the slide cool.

7. Simple Stain Simple Stains- Crystal violet and methylene blue
Place a drop(s) of stain over the smear. Make sure it covers the entire area of the smear.  Leave the stain on the smear for one minute.  Rinse with water from the bottle. 
Refer to page 64 for cellular morphology

8. Micrococcus luteus stained with methylene blue

9. Gram Stain
Gram Stain- See Gram Stain Directions on separate page.  Please refer to pages in your laboratory manual.   
All staining work is to be done at the sink
Care should be taken to work directly over the sink
Place drop(s) of crystal violet stain on the smear ( 1 minute)
Rock or roll the slide to cover the area
Use the water bottle to drip water down the slide
Place drop(s) of iodine on the slide ( 1 minute)
Place drops of alcohol on the slide 10 seconds ( KEY – do not leave on longer than 10 seconds or it will decolorize)
Place drop(s) of saffranin on the slide for 1 minute
Rinse with water from the bottle
Let the slide air dry

10. Gram Staininig examples

11. Reagents Crystal Violet (the Primary Stain)
Iodine Solution (the Mordant)
Decolorizer (ethanol is a good choice)
Safranin (the Counterstain)
Water (preferably in a squirt bottle)

12. Selective Media
Selective media is used to isolate specific groups of bacteria
They include ingredients that inhibit the growth of one type of bacterium and permit the growth of another

13. Differential Media
Specialized media assists in the identification of bacteria
Media contains ingredients that are essential for the completion of specific biochemical reactions
Assist in the identification of bacteria that are closely related by observing both the bacterial growth and the appearance of the agar

14. Mannitol Salt Agar This media contains
7.5% salt which is inhibitory to many organisms( organisms that can exist in a high salt environment are described as halophilic) Staphlococci are uniue in this respect
It also contains a carbohydrate mannitol – which is a sugar that staphylococci are capable of utilizing for energy through the process of fermentation

15. Phenol Red
Phenol red is a pH indicator for detecting the acid that is produced by fermentation of mannitol
The phenol red changes to yellow in the presence of acid.
The staphylococci exhibit this characteristic color

16. MacConkey Crystal violet is present in this agar
It inhibits the growth of Gram Positive Bacteria
It does permits the growth of Gram Negative Bacteria

17. MacConkey( continued)
It contains the sugar lactose
Bile salts
The pH indicator neutral red

18. MacConkey as Differential
This agar distinguishes between enteric bacteria on the basis of their ability to ferment lactose sugar.
If a bacterium is capable of fermenting lactose it produces pinkish colonies. Non fermenter colonies are purple

19. Eosin – Methylene Blue( EMB)
Lactose sugar
Dyes eosin and methylene blue permit differentiation between enteric lactose fermenters and non fermenters
It is also possible to identify E. coli
Because it produces a characteristic blue-black color with a metallic green sheen

20. PEA( Phenylethyl alchohol agar)
This media is used to identify gram positive bacteria
The phenyl ethyl alchohol inhibits the growth of gram negative organisms due to the construction of their cell wall.
If gram negative organisms grow their colonies are smaller than on other media