Surgery and Healing in the Developing World - Dartmouth-Hitchcock

8
64 Surgery and Healing in the Developing World
Bacteria
Important information such as shape and degree of motility can be obtained by
observation of living bacteria with the light microscope. However bacteria are routinely
stained with different dyes in order to reveal different properties and to enhance
contrast for viewing with conventional bright field microscopy. The staining
methods described kill the bacteria and prevent distortion of morphology upon
dying. Thus samples can be spread onto a glass slide and air dried, then fixed to the
surface by passing the slide quickly through a flame effectively killing the cells.
Pus, Sputum, CSF and Aspirates
The Gram Stain
The Gram stain is routinely used as an initial procedure in the identification of
an unknown bacterial species. A species can be classified as Gram positive, Gram
negative, or Gram variable depending on the ability of cells to retain the blue dye.
Gram negative bacteria do not retain the dark blue color and appear red under the
microscope. Gram positive bacterial do retain the dark blue color and thus appear
blue under the microscope.
Gram Staining Procedure
1. Prepare a heat fixed smear of the culture you wish to examine.
2. Flood the smear with crystal violet (purple basic dye) using an eyedropper
(30 sec-2 min).
3. Quickly and gently wash off excess stain with running water until the
water runs clear (2 sec).
4. Flood the smear with Gram’s iodine using an eyedropper (1min); repeat
water rinse.
5. Decolorize with alcohol (10-20 sec or until the excess alcohol which flow
off the slide is colorless).
6. Quickly and gently wash off excess stain (2 sec).
7. Flood the smear with red basic dye safranin using an eyedropper (30-120
sec).
8. Quickly and gently wash off excess stain (2 sec).
9. The smear is blotted (not wiped) to remove excess water, using bibulous
(absorbent) paper or a paper towel. Allow slide to air dry before viewing.
Microscopic Observation of a Gram Stain
Focus as described in microscopic observation of a Leishman stain with the final
step being observation under oil immersion (100X). The immersion oil is placed
directly on the smear. Higher magnifications are needed in order to see detail, as at
low magnification Gram-stained material looks like dirt on the slide. Bacteria are
often concentrated in a ring around the original smear. Bright field oil immersion
microscopy is necessary to see an undistorted image of any directly stained bacterial
smear. A Gram negative or positive phenotype cannot be confirmed with certainty
using only a dry magnification. After putting immersion oil on a slide, the high dry
(40X or 100X) lens can’t be used again unless the oil is removed. Blot slides to
remove excess oil, dip in xylene several times to dissolve the oil film, and air dry.
Note: xylene is hazardous, be sure to air dry in a well-ventilated area. Remember
Gram negative appear bright red while Gram positive appear blue under the microscope.