Preparation of Bacterial Smear

Requirements:
 24 hours culture of Bacillus cereus or Staphylococcus aureus
 Glass slides
 Bunsen burner
 inoculating loop
 needle, and glassware marking pencil

Principle: Smear preparation technique consists of spreading small volume of

sample on a slide and air drying the film before staining and microscopy.
Bacterial smears must be prepared prior to any of the staining techniques.
Purpose: to place an appropriate concentration of cells on a slide and then Simple Staining
cement them there so that they do not wash off during the subsequent staining Principle:
procedure.   The bacterial smear is stained with a single reagent, which
Step I: Preparation of the glass slide: produces a distinctive contrast between the organism and its
 Clean, grease free slides are needed for smear preparation. background. Basic stains with a positively charged chromogen are
 Grease or oil from the fingers on slides must be removed by preferred because bacterial nucleic acids and certain cell wall
washing the slides with soap and water components carry a negative charge that strongly attracts and binds
 Finally rinse the slide with 95% alcohol and dry it. to the cationic chromogen. The purpose of simple staining is to
 Hold the slide by their edge. elucidate the morphology and arrangement of bacterial cells
Step II: Labeling of slides: Materials:
 Proper labelling of the slide is essential.  Distilled H2O
 Every slide should be labelled clearly.  Bibulous paper
 A lead pencil is used to write on the frosted areas of the glass slide.  Staining tray
 Step III: Preparation of smear:  Bacterial smear
 An evenly spread smear should be prepared covering area of 15-  Acidic stain (Crystal Violet)
20mm diameter. Procedure:
 Avoid thick and dense smear because thick smear prevents light 1. Place a slide on the staining tray and flood the smear with one of the
penetration to visualize the morphology of cell. indicated stains, using the appropriate exposure time for each: 
 A good smear is one that, when dried, appears as a thin whitish  carbol fuchsin- 15 to 30 seconds
layer or film. The print of textbook should be legible through the  crystal violet- 20 to 60 seconds
smear.  methylene blue- 1 to 2 minutes
Different techniques are used for smear preparation depending upon 2. Gently wash the smear with tap water to remove excess stain. During this
culture media step, hold the slide parallel to the stream of water; in this way you can reduce
Broth cultures (liquid medium): the loss of organisms from the preparation.
 Resuspend the culture by tapping the tube with your finger. 3. Using bibulous paper, blot dry, but do not wipe the slide.
 Depending on the size of the loop, one or two loopfuls should be 4. Examine all stained slides under oil immersion.
applied to the center of the slide with a sterile inoculating loop and Result:
spread evenly over an area about the size of a dime.  Bacilli and diplobacilli: Rod-shaped bacteria, purple
 Set the smears on the laboratory table and allow to air-dry  Spirilla: spiral-shaped bacteria, purple
Culture plates (Solid medium):  Cocci: spherical-shaped, bacteria, purple
 Organisms cultured in a solid medium produce thick, dense surface
growth and are not amenable to direct transfer to the glass slide.
 These cultures must be diluted by placing one or two loopfuls of
water on the center of the slide in which the cells will be
emulsified.
 Transfer of the cells requires the use of a sterile inoculating loop or
a needle.
 Only the tip of the loop or needle should touch the culture to
prevent the transfer of too many cells.
 Suspension is accomplished by spreading the cells in a circular
motion in the drop of water with the loop or needle. This helps to
avoid cell clumping.
 The finished smear should occupy an area about the size of a
nickel and should appear as a translucent, or semitransparent,
confluent whitish film
Step IV: Air dry
 Smear should be allowed to dry completely at room temperature at
safe place
Step V: Fixation of smear:
 The purpose of fixation of smear is to preserve and prevent smear
being washed away during staining.
 Smears are fixed by heat, alcohol and occasionally by other
chemical.
Heat fixation
 After smear is air dried completely, rapidly pass the 3-4 times
through flame of Bunsen burner or sprit lamp. Negative Stain
 Avoid too much heating. Principle of Negative Staining
 After heat fix, allow the smear to cool before staining.  Since the surface of most bacterial cells is negatively charged, the
Alcohol fixation: cell surface repels the stain. The glass of the slide will stain, but
 Allow smear to air dry completely the bacterial cells will not. The bacteria will show up as clear spots
 Fix the smear with one or two drops of 70% alcohol, and leave it against a dark background.
for 2 minutes until the alcohol dries up. Purpose:  The main purpose of Negative staining is to study the
RESULT: morphological shape, size and arrangement of the bacteria cells that are
difficult to stain. eg: Spirilla. It can also be used to stain cells that are too 1. Take a clean, grease free slide.
delicate to be heat-fixed. 2. Prepare the smear of suspension on the clean slide with a loopful
of sample.
Materials: 3. Air dry and heat fix
 Negative stain (Nigrosin or Indian ink) 4. Crystal Violet was poured and kept for about 30 seconds to 1
 Bacterial culture minutes and rinsed with water.
 Inoculating loop 5. Flood the gram’s iodine for 1 minute and wash with water.
 Microscope slides 6. Then, wash with 95% alcohol or acetone for about 10-20 seconds
 Staining tray and rinse with water.
 Bunsen burner 7. Add safranin for about 1 minute and wash with water.
8. Air dry, blot dry and Observe under Microscope.
 Slide warming tray
Result:
Procedure of Negative Staining
Gram Positive: Blue/Purple Color
(doesn't used bunsen burner)
Gram Negative: Red Color
1. Place a very small drop (more than a loop full, less than a free-falling drop
from the dropper) of nigrosin near one end of a well-cleaned and flamed slide.
2. Remove a small amount of the culture from the slant with an inoculating
loop and disperse it in the drop of stain without spreading the drop.
3. Use another clean slide to spread the drop of stain containing the organism
using the following technique.
4. Rest one end of the clean slide on the center of the slide with the stain. Tilt
the clean slide toward the drop forming an acute angle and draw that slide
toward the drop until it touches the drop and causes it to spread along the edge
of the spreader slide. Maintaining a small acute angle between the slides, push
the spreader slide toward the clean end of the slide being stained dragging the
drop behind the spreader slide and producing a broad, even, thin smear.
5. Allow the smear to dry without heating. Gram Positive Bacteria: Actinomyces, Bacillus, Clostridium,
6. Focus a thin area under oil immersion and observe the unstained cells Corynebacterium, Enterococcus, Gardnerella, Lactobacillus, Listeria,
surrounded by the gray stain. Mycoplasma, Nocardia, Staphylococcus, Streptococcus, Streptomyces
Result: Gram Negative Bacteria: Escherichia coli (E. coli), Salmonella, Shigella,
and other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter,
Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella etc.

Acid Fast stain

 The acid-fast stain is a differential stain used to identify acid-fast
organisms such as members of the genus Mycobacterium. 
 Acid-fast organisms are characterized by wax-like, nearly
impermeable cell walls; they contain mycolic acid and large
amounts of fatty acids, waxes, and complex lipids.
Principle: Heat softens the wax in the cell wall and allows the stain (basic
fuchsin) to enter. The fuchsin dye is more soluble in phenol than in water or
alcohol. Phenol in turn is more soluble in lipids or waxes, thus the dye-phenol
mixture enters the cell.
Differential Staining
Procedure of Acid-Fast Stain
 In this method more than one stain is employed. In some methods
the stains are applied separately, while in other methods they are 1. Prepare bacterial smear on clean and grease free slide, using sterile
mixed and applied in one application. These procedures show technique. Allows smear to air dry and then heat fix.
differences between the cells or parts of a cell and can be used for a. Alcohol-fixation: This is recommended when the
identification. The two most important differential stains used by smear has not been prepared from sodium hypochlorite
bacteriologists are Gram stain and Acid-Fast stain. (bleach) treated sputum and will not be stained
immediately. M. tuberculosis is killed by bleach and
Gram Stain during the staining process. Heat-fixation of untreated
Principle: When the bacteria is stained with primary stain Crystal Violet and sputum will not kill M. tuberculosis whereas alcohol-
fixed by the mordant, some of the bacteria are able to retain the primary stain fixation is bactericidal.
and some are decolorized by alcohol. The cell walls of Gram-positive bacteria 2. Cover the smear with carbol fuchsin stain. Heat the stain until
have a thick layer of protein-sugar complexes called peptidoglycan and lipid vapour just begins to rise (i.e. about 60 C). Do not overheat. Allow
content is low. Decolorizing the cell causes this thick cell wall to dehydrate the heated stain to remain on the slide for 5 minutes.
and shrink, which closes the pores in the cell wall and prevents the stain from  Heating the stain: Great care must be taken when
exiting the cell. So, the ethanol cannot remove the Crystal Violet-Iodine heating the carbol fuchsin especially if staining is
complex that is bound to the thick layer of peptidoglycan of gram positive carried out over a tray or other container in which
bacteria and appears blue or purple in color. highly flammable chemicals have collected from
Mordants: Mordants are not dyes. They are important to increase the previous staining.
biological specimen’s affinity for a dye. Some stains never stain the cells or  Only a small flame should be applied under the slides
its components unless treated with a mordant. The mordant becomes attached using an ignited swab previously dampened with a few
to a cell or its components and then combines with the stain to form an drops of acid alcohol or 70% v/v ethanol or methanol.
insoluble color complex.  Do not use a large ethanol-soaked swab because this is
a fire risk.
Reagents Used in Gram Staining 3. Wash off the stain with clean water.
 Crystal Violet- the primary stain  Note: When the tap water is not clean, wash the smear
 Iodine- the mordant with filtered water or clean boiled rainwater
 acetone and alcohol (95%) decolorizer 4. Cover the smear with 3% v/v acid alcohol for 5 minutes or until
 Safranin- the counterstain the smear is sufficiently decolorized, i.e. pale pink.
Caution: Acid alcohol is flammable, therefore use it with care
Procedure of Gram Staining well away from an open flame.
5. Wash well with clean water.
 6. Cover the smear with malachite green stain for 1–2 minutes, using
the longer time when the smear is thin.
7. Wash off the stain with clean water.
8. Wipe the back of the slide clean, and place it in a draining rack for
the smear to air-dry (do not blot dry).
9. Examine the smear microscopically, using the 100 X oil immersion
objective.

Result:
 Acid fast: Bright red to intensive purple (B), Red,
straight or slightly curved rods, occurring singly or in
small groups, may appear beaded
 Non-acid fast: Blue color (A)