Using the oil immersion lens,
examine the smear for presence of bacterial cells. Note the Gram
reaction (e.g. positive or negative), morphology (e.g. cocci or
bacilli), and arrangements (e.g. single cells, pairs, clusters, or
chains) of the cells seen. This information can provide a preliminary
diagnosis regarding the infectious agent(s) and is used frequently to
direct initial therapies for the patient.
A
Gram stain of clinical material (the direct smear) should also be
examined for the presence of inflammatory cells (phagocytes or PMNs) and
epithelial cells. Inflammatory cells are indicators of an infectious
process. In respiratory specimens, the presence of epithelial cells may
be an indication of contamination with organisms and cells from the
mouth (more information on the examination and interpretation of Gram
stains of respiratory specimens will be given in Lab 4). The staining
characteristics of host cells observed in a direct smear helps in
determining if the smear has been correctly prepared and stained. Red
and white blood cells do not stain with crystal violet and will appear
pink.
Some
organisms expected to be observed in Gram stains during this course are
listed below with the expected Gram reaction, morphology, and frequent
arrangements of each.
Gram-positive organisms (stain purple)
# Gram-positive cocci
- Staphylococci -spheres occurring in irregular clusters, singly, in pairs, or in short chains.
- Streptococci – round or oval-shaped usually occurring in pairs and short, or long chains. These cocci are generally slightly elongated.
- Pneumococci –resemble streptococci. Distal ends of paired organisms may be lancet shaped. In direct smears and sometimes from a culture, a halo (capsule) may be observed.
- Yeast- large ovoid to spherical forms often occurring in clusters. Budding forms may be observed.
# Gram- positive rods
- Corynebacterium- occur as straight or slightly curved rods. They vary greatly in dimensions and can occur in parallel, or "V", "L", and "Y" arrangements. Often referred to as diphtheroids.
- Bacillus spp. (aerobic spore-formers)- large, fat bacilli occurring singly, in pairs or chains. They frequently stain gram-variable (both pink and purple). Spores generally do not stain but appear clear and can give the bacilli a "moth-eaten" appearance.
Gram-negative organisms (stain red)
- Gram-negative bacilli
Examples: Enterobacteriaceae, Pseudomonas spp. and Haemophilus spp.
The
Gram-negative bacilli often have similar characteristics in the Gram
stain; therefore, it is impossible to even presumptively identify them
strictly on the basis of stained appearance. Generally, the Haemophilus can be coccoid or bacillary (sometimes short round-ended rods referred to as a coccobacillus) and the Enterobacteriaceae and Pseudomonas spp. are larger and more elongated in shape.
- Gram-negative cocci : Spheres occurring singly and in pairs. Neisseria and Moraxella spp. often have a "coffee bean" shape where the adjacent surfaces of the pair are flattened.