Microbiology

Review of Gram StainSelective and differential Media

Gram Positive Bacteria

Staphylococcus aureus Streptococcus salivarius Bacillus subtilis Staphylococcus epidermidis

Gram Positive Cell Wall

Gram Negative Bacteria

E. coli Pseudomonas aeruginosa Proteus vulgaris Enterobacter aerogenes

Gram Negative Cell Wall

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.

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

Micrococcus luteus stained with methylene blue

Gram Stain

Gram Stain- See Gram Stain Directions on separate page.  Please refer to pages 71-73 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

Gram Staininig examples

http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gram3.htm

http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gramstains/small/tocframeset1.htm

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)

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

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

Mannitol Salt Agar

This media contains1. 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

2. It also contains a carbohydrate mannitol – which is a sugar that staphylococci are capable of utilizing for energy through the process of fermentation

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

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

MacConkey( continued)

It contains the sugar lactose Bile salts The pH indicator neutral red

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

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

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