Media and Staining

Selective Medium: culture medium that allows the growth of certain types of organisms, while inhibiting the growth of other
organisms
- dyes in the medium (e.g.: methylene blue in EMB & crystal violet in MacConkey's) or high salt concentration in the
medium (e.g.: 7% salt in MSA) inhibit the growth of unwanted microorganisms.
Differential Medium: culture medium that allows one to distinguish between or among
different microorganisms based on a difference in colony appearance (color, shape, or
growth pattern) on the medium.
- dyes in the medium (e.g.: eosin/methylene blue in EMB) or pH indicators change the color of the medium as sugars in the
medium (e.g.: lactose in EMB & MacConkey's and mannitol in MSA) are fermented to produce acid products.
 
Media Selective Inhibits Differential (+) (-)
for
EMB (Eosin Methylene Blue) Agar Selective gram-positive lactose gram-
for bacteria (e.g.: fermentation negative
gram- Staphylococc gram-negative bacteria
negative us aureus in Enterobacteria Proteus
bacteria the image Escherichia coli and vulgaris
below) is Enterobacter and
inhibited by aerogenes ferment Salmon-
the eosin & lactose (Coliform) ella typhi-
methylene murium
blue dyes in E. coli produces grow on
the media colonies with a EMB agar,
characteristic green but do not
metallic sheen on ferment
EMB agar. (Coliform) lactose
(Non
E. aerogenes coliform)
produces pink
mucoid colonies
often with a central
dark purple dot (fish
eye colonies) on EMB
agar (Coliform)
selective growth of differential for: gram-
for: gram-positive lactose negative
gram- bacteria (e.g.: fermentation bacteria
negative Staphylo- neutral red pH Salmon-
bacteria coccus aureus indicator turns red in ella typhi-
in the image presence of acid by- murium
below) is products of lactose grow on
inhibited by fermentation Mac-
the crystal Conkey's
violet dye gram-negative agar, but
and bile salts Enterobacteria do not
in the media Escherichia coli ferment
ferment lactose lactose
(media
E. coli produces appears
pink to red
yellow to
colonies often
light pink
with or without
reddish bile ppt in color &
surrounding colonies
colonies on are
MacConkey's colorless;
agar hot pink swarming
-Coliform- of Proteus
E.coli) is
inhibited
Media Selective for Inhibits Differential (+) (-)
MSA (Mannitol Salt Agar) selective 7% salt in the differential for: Staphylo-
for: gram- medium mannitol coccus
positive inhibits the fermentation epidermidis
Staphylo- growth of phenol red pH grows on
cocci most gram- indicator turns MSA, but
bacteria positive and yellow in the does not
gram-negative presence of acid ferment
bacteria by-products of mannitol
mannitol (media
fermentation remains
light pink in
Staphylococcus color &
aureus ferments colonies are
mannitol colorless
S. aureus changes
the color of the
medium from
pink to yellow
due to acid by-
products of
mannitol
fermentation
Hemolysis with Blood Selective for Inhibits most differential for: hemolysis... gamma-
Gram positive gram negative particularly in streptococci hemolysis (no
Agar (Staph, Strep, based on the ability to break hemolysis):
agar contains 5% sheep's and Colistin down hemoglobin or red no hemolysis
blood Enterococcus) Affects blood cells, 3 groups of is observed;
membrane microorganisms can be bacteria do
CNA (Columbia ) Colistin integrity described not produce a
Nalidixic Acid alpha-hemolysis: a green to hemolysin
Nalidixic Acid light-brown halo is seen
Interferes with around the colonies; bacteria Does not
DNA partially break down hemolyze
replication hemoglobin leaving a green RBCs.
(Klebsiella, pigment (biliverdin) α ( gamma
Proteus, hemolytic (Staph, Strep or hemolytic
Pseudomonas) Enterococcus) Staph, Strep
beta-hemolysis: a clearing is or
seen around the colonies; Enterococcus)
bacteria produce a "beta-
hemolysin" (streptolysin O or
Green color for biliverdin, the partial S), which lyses red blood cells
breakdown of blood; in our bodies. in the medium: β hemolytic
(Staph, Strep or
Enterococcus)
Hektoen Enteric Agar Gram Negative Bile Salts Blue Green with black ppt Blue Green
will grow. inhibit most does not ferment lactose. with no black
gram positive Reduces sulfur to hydrogen ppt. (does not
cocci. Dyes sulfide (H₂S) (Salmonella) ferment
like lactose, does
bromthymol Yellow to Salmon Pink not reduce
blue and acid Acid from fermentation sulfur) (No
fushsin inhibit (lactose) Coliform (E. coli) Reaction)
growth (Shigella)
Media Selective for Inhibits Differential (+) (-)
Phenylethyl Alcohol Agar Undefined that allows Inhibits
growth of Gram positive most Gram
Phenylethyl alcohol interferes with
organisms negative
DNA synthesis in gram negative Isolates staphylococci and organisms
organisms streptococci (including (E. coli and
enterococci and lactococci) Proteus)
Oxidation Fermentation Oxidation No sugar Metabolism
Any amount of Yellow Green or blue (nonsaccharolytic)
Test Does not break down sugars or
Oxidation or starches to monomers (glucose)
Fermentation
Add iodine to turn black. Yellow throughout

Amylase breaks down starch Slightly yellow at top

Amylose to Glucose Oxidation and slow
fermentation or slow
fermentation only

Starch Hydrolysis Test Clearing around growth No clearing around growth

Amylase digests starch Amylase is not present to digest
the starch

Starch (Amylase) broken up into

monomers of carbohydrates
which is glucose.
Reagent is Iodine
Casein Hydrolysis Test Clearing in agar (Milk No clearing in the agar
Agar) – Casease is present Casease is absent. (E. coli)
No Reagent is used Proteins broken down to
Protealytic (Proteins broken amino acids (B. cereus)
down into monomers called
amino acids)
Lipid Hydrolysis Test Clearing in agar and No clearing in the agar Lipase is
around growth- Lipase is absent.
Peptone present. Lipids are
Lipids (TG) broken up into broken down into
monomers which are (fatty acids monomers of glycerol
and glycerol to release carbon and fatty acids.
energy)
Media Selective for Inhibits Differential (+) (-)
Fluid Thioglycollate Medium (liquid) Grow at the top pH oxidation/
(aerobic) reduction
Oxygen utilization.
Pseudomonas
Aerotolerance
aeruginosa (and Aerobic Zone
Obligate (strict) aerobes
Yeast); indicated by
Obligate (strict) anaerobes
Grow on the bottom Resazurin (pink
Facultative anaerobes (E. coli)
(anaerobic) color) on top
Capnophiles-CO₂ levels elevated
Clostridium (oxidized).
sporogenes
Casein
Facultative anaerobes Anaerobic Zone
grow throughout: indicated by
Staphylococcus straw colored,
aureus reduced.
Temperature 5⁰ 25⁰ C pink color
Serratia
Phenotype-genetic expression. Best psychrophiles marcesens
temperature to not affect the 25⁰
proteins. mesophiles
Escherichia coli
Trypticase Soy Agar (TSA) 37⁰
mesophiles
60⁰
Trypsinogen is enzyme.
thermophiles
pH effect on microbial growth Acidophiles
(1-6)
Yeast
Alkaliphiles
(8-14)
Neutrophiles
(7)

Shapes Single Two 4 8 Cube Chain Cluster

Perpendicula
r
Round Cocci Coccus Diplo Tetrad Sarcina Strepto Staphylo
Short Rod Coccobacilli
Rod Bacilli Bacillus Diplo Tetrad Sarcina Strepto
Curved Rod Vibrios
Spiral Spirilla Spirillum
Flexible Spirochetes
spirals
Dry, heat fix then stain.

Gram staining consists of four components:

Primary stain (Crystal violet) 1 minute then water
Mordant (Gram's Iodine) 1 minute then water
Decolourizer (ethyl alcohol) until runoff clear then water
Counterstain (safranin ) 1 minute then water

The slide is washed in water; excess water is removed using a blotting paper.
Those bacteria that hold on to primary dyeiodine complex and remain violet are called
Gram positive and those which get decolorized and subsequently take up
counterstain (pink/red) are called Gram negative.
Basic fuchsin (present in dilute carbol fuchsin) stains many Gram negative bacteria
more intensely than does safranin, making them easier to see. Some bacteria which are
poorly stained by safranin, such as Haemophilus spp., Legionella spp., and some
anaerobic bacteria, are readily stained by basic fuchsin. It is believed that the positively
charged crystal violet pass through the cell wall and cell membrane and binds to
negatively charged components inside the cell. Addition of negatively charged iodine (in
the mordant) binds to the positively charged dye and forms a large dye-iodine complex
within the cell. Crystal violet (hexamethyl-para-rosaniline 3chloride) interacts with
aqueous KI-I2 via a simple anion exchange to produce a chemical precipitate. The small
chloride anion is replaced by the bulkier iodide, and the complex thus formed becomes
insoluble in water. During decolorization, alcohol dissolves the lipid present in the outer
membrane of Gram negative bacteria and it leaches the dye-iodine complex out of the
cell. A thin layer of peptidoglycan does not offer much resistance either. The dye-iodine
complexes are washed from the Gram negative cell along with the outer membrane.
Hence Gram negative cells readily get decolorized. On the other hand Gram positive
cells become dehydrated from the ethanol treatment, closing the pores as the cell wall
shrinks during dehydration. The dyeiodine complex gets trapped inside the thick
peptidoglycan layer and does not get decolorized.