Professional Documents
Culture Documents
A Presentation By
G. Prashanth Kumar
Department of Microbiology & Parasitology,
Faculty of Medicine, International Medical & Technological University,
Dar-Es-Salaam, Tanzania.
INTRODUCTION:
Bacteria are microscopic organisms. They are also colorless for the
STAIN:
POSITIVE STAINING: - where the actual cells are themselves colored and appear
in a clear background.
(a) Simple staining: A stain which provides color contrast but gives same color
to all bacteria and cells.
Ex: Loeffler’s methylene blue, Polychrome methylene blue, Diluted carbol
fuchsin.
(b) Nigrosin .
BACTERIAL SMEAR PREPARATION:
Method:
-Aseptically a small sample of the culture is spread over a slide surface.
-The next step is heat fixation to help the cells adhere to the slide surface.
The paraffin is first removed with xylene, the xylene is then removed with
alcohol and the alcohol is replaced with water.
2) Methanol fixation
a) Place air-dried smears in a coplin jar with methanol for one minute.
Alternatively, flood smear with methanol for 1 minute.
The slow oxidation of the methylene blue forms a violet compound that gives
the stain its polychrome properties.
The ripening takes 12 months or more to complete, or it may be ripened
quickly by the addition of 1.0% potassium carbonate (K2co3) to the stain.
Incontrast to the blue staining of most structures by the methylene blue, the
violet component stains acidic cell structures red-purple , e.g. the acid capsular
material of the anthrax bacillus in the McFadyean reaction.
Made by diluting Ziehl-Neelsen’s stain with 10-20 times its volume of water.
Stain for 10-25 seconds and wash well with water. Over-staining must be
avoided, as this is an intense stain, and prolonged application colours the cell
protoplasm in addition to nuclei and bacteria.
REQUIREMENTS
Distilled Water
Compound Microscope
Fixed smear
PROCEDURE
Heat fixes the smear by passing the slide 2-3 times gently over the Bunsen
flame with the smear side up
Pour Loeffler’s methylene blue over the smear and allow it to stand for 3
minutes.
Wash the stained smear with water and air dry it.
Observe the smear first under low power (10x) objective, and then under oil
immersion (100x) objective.
Observe the presence of organisms and also the cellular content of sample.
SIMPLE STAINING: LOEFFLERS METHYLENE BLUE
GRAM STAINING
Gram positive.
Gram negative.
HANS CHRISTIAN JOACHIM GRAM
16
ORIGINAL FORMULATION OF DR. GRAM
Lugol’s Iodine,
Absolute Alcohol,
Bismarck Brown
CARL WEIGERT (1845-1904)
German pathologist Carl Weigert (1845-1904) from
Frankfurt, added a final step of staining with
safranin.
18
PRINCIPLE
The Gram Reaction is dependent on permeability of the bacterial cell wall and
cytoplasmic membrane, to the dye-iodine complex.
In Gram positive bacteria, the crystal violet dye –iodine complex combines to form
a larger molecule which precipitates within the cell. Also the alcohol/acetone
mixture which act as decolorizing agent, cause dehydration of the multi-layared
peptidoglycan of the cell wall. This causes decreasing of the space between the
molecules causing the cell wall to trap the crystal violet iodine complex within the
cell. Hence the Gram positive bacteria do not get decolorized and retain primary
dye appearing violet. Also, Gram positive bacteria have more acidic protoplasm and
hence bind to the basic dye more firmly.
In the case of Gram negative bacteria, the alcohol, being a lipid solvent, dissolves
the outer lipopolysaccharide membrane of the cell wall and also damage the
cytoplasmic membrane to which the peptidoglycan is attached. As a result, the
dye-iodine complex is not retained within the cell and permeates out of it during
the process of decolourisation. Hence when a counter stain is added, they take up
the colour of the stain and appear pink.
GRAM POSITIVE BACTERIA
22
METHOD CONSISTS OF FOUR COMPONENTS:
Primary staining: The smear is covered with gentian violet, for 1 minute and
washed with water.
Mordanting: It is then covered with Gram’s iodine, Kept for 1 minute, and washed
with water.
Decolourisation: The smear is covered with alcohol and is washed with water
immediately.
Counter staining: The smear is then covered with safranine, kept for 30 seconds
and washed with water.
Using filter paper the slide is gently blotted to dry. Place a drop of cedar wood
oil/Liquid paraffin on the smear.
Adjust the microscope for increased light by raising the condenser, and the slide is
RESULT:
26
PROCEDURE FOR TISSUE SECTION
Result:
Gram-positive organisms, fibrin, some fungi, keratohyalin, and keratin - purple
Gram – negative organisms -red.
27
QUICK GRAM METHOD
By this method fairly good results may be obtained with very short staining
times, which are convenient when only one slide has to be stained.
Flood the slide with crystal or methyl violet stain and allow to act for about 5
seconds.
Tip off the stain and flood the tilted slide with iodine solution and allow to act
for about 5 seconds.
Tip off the iodine and flood the tilted slide
With acetone and allow this to act for only 2 seconds before washing it off with
water from the tap.
Flood the slide with basic fuchsin counter stain and allow it to act for about 5
seconds. Wash off with water, blot and dry.
QUALITY CONTROL
Daily and when a new lot is used, prepare a smear of Escherichia coli (ATCC
25922) and Staphylococcus epidermidis (ATCC 12228)or Staphylococcus aureus
(ATCC 25923). Fix and stain as described.
29
MODIFICATION IN GRAM STAINING METHODS
Bartholomew (1962) has pointed out that each variation in the Gram
staining procedure has a definite limit to its acceptability. Any final
result is the outcome of the interaction of all of the possible
variables.
Prolonged decolourization
Excessive counterstaining
32
APPLICATIONS OF GRAM STAINING
5.Counting of bacteria.
33
ZIEHL-NEELSEN STAINING FOR ACID FAST BACILLI
The Ziehl-Neelsen acid fast staining method has proved to be most useful for
staining acid fast bacilli belonging to the genus Mycobacterium especially
Mycobacterium tuberculosis and Mycobacterium leprae, and also for Nocardia.
PRINCIPLE OF ZIEHL–NEELSEN STAIN
Acid fastness of acid-fast bacilli is attributed to the presence of large quantities of
unsaponifiable wax fraction called mycolic acid in their cell wall and also the
intactness of the cell wall . The degree of acid fastness varies in different
bacteria.
In this staining method, application of heat helps the dye to penetrate the
tubercle bacillus.
Once stained, the stain cannot be easily removed. The tubercle bacilli resist the
decolorizing action of acid-alcohol which confers acid fastness to the bacteria.
Modifications :
Kinyoun’s-cold stain
ACID - FAST STAIN BASIC REQUIREMENTS
2. Decolourization with Acid Alcohol : The acid alcohol contains 3% HCl and
95% ethanol or 20% H2 SO4.
9. Add Loeffler’s Methylene Blue stain (counter stain). Leave Loeffler’s Blue
stain on smear for 15-20 seconds .
Place the dried, heat-fixed smear on a staining rack over the sink. Smears of
sputum should be thin.
Cover the smear with auramine phenol and leave to stain at room temperature
for 10 minutes.
Wash off stain with water from the tap.
Cover the slide with an excess of 1% acid-alcohol and leave to decolorize for 5
minute.
Wash off decolourizer with water from the tap.
Cover the smear with the 0.1% potassium permanganate counter stain
and leave for 15 seconds.
A sputum smear
stained with
Auramine
FLUOROCHROME AFB MICROSCOPY
More rapid and sensitive
Advantages:
• More accurate: 10% more sensitive than light microscopy, with specificity
comparable to ZN staining
Disadvantages:
Cover slide with Albert’s stain and allow to act for 3-5 min.
Cover slide with Albert’s iodine and allow to act for 1 minute.
Corynebacterium
diphtheriae Stained by
Albert’s stain Green
colored bacilli showing
"Chinese-letter"
arrangement at angles to
each other containing
bluish-black
metachromatic granules
of cells.
STAINING OF SPORES
Place the slide over a beaker of boiling water, resting it on the rim with the
bacterial film uppermost.
When, within several seconds, large droplets have condensed on the underside of
the slide, flood it with 5% aqueous solution of malachite green and leave to act for
1 min while the water continues to boil.
This method colours the spores green and the vegetative bacilli red. Lipid granules
are unstained.
MALACHITE GREEN STAIN FOR SPORES
PRINCIPLE:
Capsule staining is more difficult than other types of differential staining procedures
because the capsular materials are water soluble and may be dislodged and removed
with vigorous washing.
Bacterial smears should not be heated, because the resultant cell shrinkage may create
a clear zone around the organism, an artifact that can be mistake for capsule.
The capsule is non-ionic, so that the dyes commonly used will not bind to it. Two dyes,
one acidic and one basic, are used to stain the background and the cell wall,
respectively.
METHODS:
1) Negative staining.
2) Positive staining.
Capsulated Bacteria:
Make a smear from colony of S.pneumoniae on a clean grease free glass slide,
and allow it to air dry. Note: The smear should not be heat fixed.
Put the smear on a slide rack and flood smear with crystal violet. Allow it to stain
for 5-7 minutes.
Wash the smear with 20% copper sulphate solution and blot it dry.
Observe the smear first under low power (10x) objective, and then under oil
immersion(100x) objective.
In the culture smear, the capsule is seen as a light blue in contrast to the deep
purple colour of the cell.
For negative staining of smears:
Leptospires from a
culture stained by
modified Fontana
silver technique
(3.000×)
examination by
dark- field
microscopy.
FLAGELLA STAINING
Procedure:
After 5-10 min, when many bacteria have attached to the surfaces of
the slide and cover-slip, apply two drops of Ryu’s stain to the edge of
the cover-slip and leave the stain to diffuse into the film. Examine with
the microscope after standing 5-15 min at ambient temperature.
FLAGELLA STAIN BY RYU STAIN: MICROSCOPY
Touch the adhesive side of the tape of transparent scotch tapes on the
surface of the colony at a point intermediate between its centre and
periphery.
Fix the adhesive side of the tape over an area on the glass slide
containing the LPCB.
Remove a small portion of the colony and the supporting agar at a point
between the centre and periphery and place it in the drop of LPCB.
With a needle, tease the fungal culture first and spread in the LPCB.
Examine microscopically after giving sufficient time for the structures to take
up the stain usually 30 mins.
LACTOPHENOL COTTON BLUE: MICROSCOPY
PERIODIC ACID –SCHIFF
(METHOD FOR FUNGI IN TISSUE SECTIONS)
2)Treat for 5 min with a freshly prepared 1% solution of periodic acid in water.
5)Wash two or three times with sulphite wash solution . Wash with water.
7)Counter stain with dilute aqueous malachite green or with 0.1% light green
in 90% alcohol for 1 min.