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Microbio Lab 1 and 2 u 2006

Microbio Lab 1 and 2 u 2006

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MICROBIOLOGY LAB 1 and 2 \u2013 Staining
USTMED \u201907 Sec C \u2013 AsM; Photos provided by JV.N. & A.M.
USTNotesGroup2009

Updated 2006 - A. Abad
Stain \u2013 coloring of organisms with a dye that emphasizes
certain structures
Smear \u2013 a thin film of material containing the microorganisms
spread over the surface of the slide
Fixation \u2013 a procedure done before staining a smear to:
1. attach the microorganisms to the slide
2. kill the microorganisms
3. preserve the various parts of microbes in their
natural state with only minimal distortion
3 Methods of Fixation

1. Air drying
2. Passing over flame of Bunsen burner
3. cover slide with methyl alcohol for 1 minute

-
if not fixed, stain may wash the microbes
Stains \u2013 salts composed of a positive and a negative ion
1.basic dyes (chromatophore) \u2013 positive ion
2.acidic dyes \u2013 negative ion
Bacteria \u2013 slightly negative charge; pH 7
Add (+) ion [basic dye] to charge bacteria = attract
Ex. Crystal violet, methylene blue, safranin
Add (-) ion [acidic dye] to charge bacteria = repel
Ex. Eosin, nigrosin, acid fuchsin
Negative stain \u2013 prepare colorless bacteria against a colored
background
Importance:
1. valuable in the observation of overall shapes,
sizes and capsules

2. distortion of cell size and shape are minimized
a. heat fixing is not necessary
b. cells do not pick up the stain

Types of stain
1. Simple stain
-
Purpose \u2013 cellular shape and structure is made
visible
-
Ex. Methylene blue, carbol fuchsin, crystal violet,
safranin
2. Differential stain
-
Distinguishes two groups of organisms or different
parts of a bacterial cell
-
Ex.
a. Gram stain
b. Acid fast stain
i. Ziehl Neelsen stain
ii. Modified Kinyoun stain
c. Spore stain
i.Schaefer Fulton method
ii.Wirtz Conklin method
Reagents in Differential stain
1. Primary Stain \u2013 imparts color to all cells
-
Ex.
Gram stain

Crystal violet
Acid fast stain Carbol fuchsin
Spore stain

Malachite green
2. Mordant \u2013 a chemical added to primary stain to:
a. Intensify
b. Increase affinity
c. To coat a structure to make it thicker
- Types of mordant
o
Physical mordant \u2013 steaming
o
Ex.

Ziehl Neelsen stain
Schaefer Fulton method
Wirtz Conklin method

o
Chemical mordant
o
Ex.
Gram stain
Grams iodine
Flagellar stain
Tannic acid
Modified kinyoun stain phenol
3. Decolorizer \u2013 a chemical to remove the primary stain
- Ex.
Gram Stain
95% C2H2OH
(acetone alcohol)
Acid fast stain Acid alcohol
4. Secondary stain \u2013 a chemical which imparts a
contrasting color to the primary stain
- Ex.
Gram stain Safranin
Acid fast
Methylene blue,
malachite green
Spore
stain
safranin
I. Gram Stain
Purpose
Reagents
Gram +
Gram -
Primary
stain
Crystal violet Violet or
purple
Violet or
purple
Chemical
Mordant
Grams iodine Dark violet
or purple
Dark violet
or purple
Decolorizer
Acetone
Alcohol
Violet or
purple
Colorless
Secondary
stain
Safranin
Violet or
purple
Red
End Result
Violet
Red
Gram stain of Micrococcus
species.

Microscopically, micrococci are larger than staphylococci and appear in tetrads

rather
than
grapelike clusters.
Urethral discharge with PMN
and
intracellular
gram
negative
diplococci
suggestive
of
Neisseria
gonorrhoeae.
Principle:
1. When applied to both gram-positive and gram negative cells,
crystal violet and then iodine readily enter the cells.

2. Inside the cells, the crystal violet and iodine combine to fome CV-1 which is larger than the crystal violet molecule that entered the cells.

3.In Gram positive cells, crystal violet-iodine complex is
trapped and therefore, retains the color.
4.In Gram negative cells, the alcohol disrupts the
lipopolysaccharides and the CV-I complex is washed out
through the thin peptidoglycan layer.
5. Gram negative cells are colorless until counterstained with
safranin after which they are pink.
Clinical Significance

- Provides valuable information for the treatment of disease
Gram
+

Penicillin
Cephalosporin
Gram
-
More resistant because the antibiotics do not
penetrate the lipopolysaccharides
II. Acid Fast Stain
Purpose
Reagents
Gram +
Gram -
Primary
stain
Carbol
fuchsin
Red
Red
Mordant
Steam or
phenol
Red
Red
Decolorizer
Acid alcohol
Red
Colorless
Secondary
stain

Methylene
blue or
malachite
green

Red
Blue or green
Modified Kinyoun acid-fast
stain.
Nocardia
species
appear
acid-fast

when stained with a modified Kinyoun stain using 2% H2SO4 as the decolorizing agent. This

feature
helps
to
distinguish

this mircroorganism from other actinomycetes.

Prinicple

1. Acid fast organisms retain the red color because the
carbol fuchsin is more soluble in the cell wall lipids than
in the acid alcohol.

2. Non-acid fast organisms \u2013 cell walls lack the lipid
components; carbol fuchsin is rapidly removed during
decolorization

Mycolic acid \u2013 a cell wall component responsible for the
acid fastness of the organism
Acid fast bacilli \u2013 organisms which are hard to stain but
once stained, they are hard to decolorize
Ex. Genus Mycobacteria, Genus Nocardia
Clinical significance
-

the finding of acid fast bacilli in a sputum is a
presumptive evidence that the organisms is
Mycobacterium tuberculosis.

III. Special stains or selective stains \u2013 stains used to color
and isolate specific parts of microorganisms such as
endospores, flagella, capsules and metachromatic granules
A. Negative staining for capsules
1. India ink technique
- mix the bacteria in a solution containing a fine
colloidal suspension of colored particles.
- then stain with a simple stain, safranin.
- Result \u2013 Halos surrounding each stained bacterial
cell.
Capsule stain. The cell is the purple

rod in the center of the clear area. The purple color is from the basic stain, crystal violet.

The clear area is the capsule, and the background is colored by the negative, acidic stain (India ink).

B. Flagella stain \u2013 a tedious and delicate staining procedure using a mordant and the stain carbol fuchsin to build up the diameters of the flagella until they become visible.

Vibrio cholerae.
Monotrichous flagella \u2013 one
polar flagellum
Proteus.
Peritrichous flagella \u2013 flagella
over the entire bacterial cell
C. Endospore (spore) staining \u2013 both selective and
differential
-
Ex. Schaefer Fulton method, Wirtz Conklin method
Purpose
Reagent
Spores Vegetative cell
Primary
stain
Malachite
green
Green
Green
Mordant
Steam
Green
Green
Rinse
Tap water
Green
Colorless
Secondary
stain
safranin
green
red

Spore stain of Bacillus cereus. The arrows are pointed at green spores in a pink vegetative cell.

Gram Stain
Purpose
Reagent
Spores
Vegetative cell
Primary stain
crystal
violet
Violet or
purple
Violet or purple
Mordant
Grams
iodine
Dark violet
Dark violet or
purple
Decolorizer
95%
C2H2OH
Colorless
Violet or purple
Secondary
stain
Safranin
red
colorless
Violet or purple

Gram stain of Bacillus cereus.
The arrow is pointed at a
spore, which is clear inside
the gram-positive vegetative
cell.

D. Metachromatic granules
-
Ex. Neisser stain, Loefflers methylene blue stain
Loefflers methylene blue stain.
Corynebacterium diptheriae
Demonstrate

metachromatic granules when stained with Loeffler methylene blue stain or Neisser stain. The stain is best performed on colonies grown on a

Loeffler
agar
slant.
Metachromatic
deposits
are
reddish
purple
in
Loeffler
methylene blue stain.
- fin -
Demo slides\u2026..
Vibrio spp. (curved bacilli)
Gram + stain
Streptococcus lactis (cocci in chains)
Sarcinae lutea (cocci in tetrads)
Congo red staining

India Ink
Malachite green staining
S. aureus
Bacillus subtillis (gram positive bacilli in chains)
Streptococcus lactis (cocci in chains)
The Gram Stain Introduction

Gram\u2019s Stain is a widely used method of staining bacteria as
an aid to their identification. It was originally devised by
Hans Christian Joachim Gram, a Danish doctor.

Gram\u2019s stain differentiates between two major cell wall
types. Bacterial species with walls containing small amounts
of peptidoglycan and, characteristically, lipopolysaccharide,
are Gram-negative whereas bacteria with walls containing
relatively large amounts of peptidoglycan and no
lipopolysaccharide are Gram-positive.

It\u2019s a mystery

Although it may seem strange, the reason why bacteria with these two major types of bacteria cell walls react differently with Gram\u2019s stain appears to be unconnected with the wall

structure itself. The exact mechanism of the staining reaction is
not fully understood, however, this does not detract from its
usefulness.

The Gram staining method

1. A small sample of a bacterial culture is removed from a culture. In this example it is being taken from a broth culture of the pure microbe but it could be removed from a culture on solid medium or from material containing bacteria eg faeces or soil.

2. The bacterial suspension is smeared unto a clean glass slide. If the bacteria have been removed from a culture or a solid media or it is from a soil or feces sample It will have to be mixed with a drop of bacteria-free saline solution

3. The bacterial smear is then dried slowly at first and the, when dry, heated for a few seconds to the point when the glass slide is too hot to handle. This fixes ie kills the bacteria making the slide safe to handle.

Care must be taken not to
overheat which will char the cells.

4. Once cool, the slide is transferred to a support over a sink and flooded with a stain called Gentian Violet (a dye consisting of a methyl derivative of pararosaniline). The stain is left on the slide for about 1 minute. This stains all the bacteria on the slide a dark purple colour. Note, this stain will not penetrate the waxy cell walls of some bacteria eg mycobacteria.

5. The Gentian Violet is gently washed off
the slide with running water

6.The bacterial smear is then treated with Gram\u2019s solution which consists of 1 part iodine, 2 parts potassium iodide, and 300 parts water. This iodine solution reacts with the Gentian Violet turning it a very dark shade of blue. It also causes it to be retained by certain types of bacteria in a way which is not really understood.

7.After about 30 seconds the slide is gently rinsed with ethyl alcohol which causes the dye-iodine complex to be washed out of some bacteria but not others. This is called decolourisation.

If we now look at the smear down a microscope, the bacteria which had retained the Gentian Violet-iodine complex will appear blue-black.

These are called Gram-positive. However wi would not be able to see those which had lost the dye-iodine complex which are called Gram-negative. The final step in the gram stain method is, therefore, to stain the Gram-negative cells so they can be seen.

8. This is achieved by treating the smear with a compound which stains the Gram- negative cells a colour which contrasts markedly with the blue-black colour of the gram-posiitve cells. The stain common used for this is either eosin or fuchsin, both of which are red. These are called counterstains.

Bacteria in the smear which are Gram-positive are unaffected by the counterstain.

9. The counterstain is left on the smear for about 30-60 seconds and then gently rinsed away with running water.

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