Unit 4

Preparation of Microorganisms for Microscopic Examination

Two major methods:

1. General characteristics:
a) Living microorganisms thus motility and morphology easily observed
b) Morphology less distorted
c) Quick and fast (not suitable for lengthy examination as it will easily dry
d) Used microorganisms not readily stained and readily cultivated
e) Used to study larger microorganisms like protozoans, molds, yeasts and unicellular algae
2. General types
a) Wet mount technique:
 Place a drop of liquid culture or from broth or portion of a colony from a solid
medium on the center of the slide
o Two approaches
 Place a loopful of liquid clinical specimen or culture on a clean glass slide and cover
it with cover glass. Observe under low or high power objective
 Place 2 or 3 drops of cell suspension onto a clean microscopic slide
 Scrape coverslip over petrolatum (thin layer on hand) to produce even edge
of material around all slides
 Place coverslip over cell suspension and press gently to distribute and seal
Note: if dark-field microscope is used the organisms would appear bright
against a dark backround.
b) Hanging fluid technique: demonstrates motility of living organisms in fluid and for a long
time
 Procedure: (use a depression slide)
 Apply a thin rim of petroleum jelly just outside the edge of the depression in a
special hollowed-out glass slide.
 A small drop of culture is placed exactly in the center of the clean cover slip.
 Slide is inverted over the coverslip so that the drop is just in the, below the hollow.
 Gentle pressure spreads the petroleum jelly and sticks the coverslip, creating a
sealed chamber.
 Entire preparation is inverted, thus the drops hangs down into the chamber
 Completed preparation can be examined under oil immersion

Microbial staining technique (non-living; fixed state)

Living microorganisms particularly bacteria are almost colorless thus the need to be stained.

1. Purpose of staining
a) For greater visibility of size and shape.
b) To differentiate cellular components and internal structures like granules and spores to aid
in identification of species.
c) For greater contrast between cells and the surrounding background.
2. Factors which determine which stain is taken up by the cell
 a) Chemical make-up of microbial cell (stain cellular components)
b) pH of surrounding material which maybe basic or acidic
3. characteristics of stain:
a) generally salts in which one of the ions is colored (either cation or anion)
4. characteristics of stains: basic, acidic, neutral
a) in basic dyes, the color of the positive ion (cation)
 composition: colored cation (+) with a colorless anion
Note: generally used because most organisms are negatively charged
 acidic components of the cell like nucleic acids, negatively charged phosphate
groups and acidic polysaccharides combine with the positively charged cation of
basic stain
 environment or medium near neutrality or slightly acidic combine with positively
charged cations of basic stains.
 Examples of basic stains
Methylene blue (30 to 60 sec.)
Crystal violet (10 sec.)
Thionin
Safranin
Neutral red
Hematoxylin
b) In acidic dyes, the color is in the negative (-) ion (anion)
 Composition: colored anion (-) with a colorless cation
 Basic components of the cell like positively charged colored anion of acidic stain
 Examples of acidic stains
Malachite green
Janus green
Acid fuchsin
Eosin
Methylene orange
Nigrosin and India ink (negative staining)
Note: acid dyes are repelled by the overall negative charge of bacterium
Result: only the background is colored
Effect: bacterial cell is coloreless against a colored background thus useful in
observing the shape of the cell
 Procedure in negative (indirect) straining
 Place the small drop of nigrosin on one end of the slide
 Mix organisms in drop
 Touch spreader slide against the drop of suspended microorganisms to
spread the drop along the edge
 Move spreader slide away from drop so that the stain and organisms are
spread over the slide
 Air dry and examine under the microscope
c) Neutral dyes:
Examples: Giemsa
 Wright
Leishman

Preparation of bacterial smear

1. Place 1 or 2 loopfuls of microorganisms suspension on the slide

Result: smear or a thin layer of cells that has been spread over a small area of the slide
2. Air dry the smear at room temperature
3. Fix smear by rapidly passing through alcohol flame 3 of 4 times
Note: Overheating causes distortion of the microorganisms
Purpose of fixation: the heat will result to coagulation of protoplasm and adherence to the slide
so as not to fall off in staining and washing
Types of microbial staining
1. Simple direct staining (quick)
a) Characteristics
 Use only one dye and one color is obtained, the color of the stain
 Obtain contrast of specimen against the background
 General morphology
b) Procedure:
 Apply stain to the fixed smear
 Stains that can be used: gentian, crystal violet (10 sec.) safranin, methylene
blue (30 sec.) basic fuchsin (5 sec.) and other aniline dyes
 Stain by flushing of flooding
 Wash of smear with a gentle stream with a gentle steam of cool water until no color
is obtained from the water. Avoid flushing or squirting water directly on the smear.
 Blot dry the smear
 Drop Canada balsam on smear and cover with a cover slip
 Observe under oil immersion
2. Differentiate staining cells exposed to more than one dye or stain which due to differences
in chemical constituents, mixed organisms can be stained and distinguished by their
different staining patterns
a) Gram stain: differentiates gram (+) from gram (-) bacteria, the two physiological groups
of all true bacteria
 Steps in gram staining
 Smear with cells in flooded with gentian or crystal violet, a basic primary
stain for 1 minute
Result: All bacteria are stained blue or violet
 Wash or flush off the stain with water until no color is traced in the
water
 Add iodine solution, a mordant (chemical that forms and insoluble
complex with dyes: CV-I complex) for 1 minute
Result: specimen or all remains blue or violet
 Wash off the iodine with water
Result: all cells remain blue black
 Wash of decolorizer , 95% ethyl alcohol or acetone- alcohol mixture for
15-30 seconds until no color washes out of the smear
Result: Grain (+) bacterial cells (with thick