Microbiology
Review of Gram Stain
Selective 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/antibi
otic_manual/Gram3.htm
http://www.uphs.upenn.edu/bugdrug/antibi
otic_manual/Gramstains/small/tocframese
t1.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 contains
1. 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