Uses / Clinical Applications
1. The gram stain is the single most useful staining procedure for the rapid presumptive ID of bacteria clinically as the basis of the preliminary report for the C & S.
2. Suggests possible identify based on gram reaction, morphology and arrangement. Gram negative diplococci (neisseria). Gram positive cocci (staphylococcus). Gram negative rod (e.coli, psuedomonas). You can’t tell e. coli apart from pseudomonas but it may not be necessary to refer treatment because certain antibiotics are known to be effective against gram negative bacteria.
3. If a gram stain is ordered STAT it could take 30-40 minutes as opposed to 12-24 hours.
4. Provides preliminary data on susceptibility to antibiotics for treatment. If the bacteria are still there after antibiotic was given, then it didn’t work.
5. Suggests media, incubation conditions for growth and isolation of bacteria from clinical specimens. For example, if you had a bite wound that suggested anaerobic bacteria, would you have to tell the lab you suspect anaerobic bacteria? Yes. Because if you set it up aerobically, the results will be meaningless. Clinical labs usually have standard operating procedures to eliminate these issues and maximize recovery. When you take a blood culture you take two cultures, one anaerobic and one aerobic.
6. May explain failure to obtain viable organisms on culture possible due to inadequate growth media and or incubation conditions: Anaerobes incubated aerobically.
Advantages: rapid, cost effective, potentially life saving.
The gram stain procedure
Gram positive bacteria have a lot of peptidoglycan (which is part of the bacterial cell) and a tight/dense matrix to retain the primary stain. If you look on page 85 of your text book, it shows you the 3 types of cell walls.  The crystal violet is going to be retained by the thick dense matrix within the peptidoglycan. When you add the alcohol, the dye remains because it’s bound firmly to the cell wall because of the thick matrix. Imagine you have a piece of net, imagine it is just one layer vesus several layers of net. Imagine you pour sand through the multiple-net layer, the sand will stay because of all the peptidoglycan.  If the matrix is loose, the primary dye is not retained. You add iodine which increases the affinity for the dye to attract to the cell wall.
When you add the alcohol, the primary crystal violet dye is washed out in a gram negative bacteria but remains in the gram positive bacteria.  At this stage the gram positive bacteria will be purple and the gram negative will be colorless.
When you add safranin (red dye) what happens to the gram positive? It will remain purple but the gram negative will become red. So you end up with gram negative that is pink/red or gram positive that is purple. (This is shown on page 59.)
Role of gram stain in C & S
The gram stain is used as the basis for treatment and is potentially life saving in critically ill patients with meningitis or sepsis. A clinician cannot wait for the complete work up of a C & S (3-5 days) to treat someone in septic shock. The gram stain could be done in 30 minutes stat or typically within 2-4 hours. The gram stain is part of the critical values data set and it’s called critical because it’s pivotal/essential/potentially-life-saving.
Role of acid fast stain in C & S
Relatively few bacteria are acid fast bacteria but it can be useful to form basis for tuberculosis or leprosy. The two genera that are AFB are actinomycetes and mycobacterium. By far the most common genus is mycobacteria and the most common bacteria are M. tuberculosis and the other is M. leprae. There are others such as M. Avium intracellulare and M. chelonae and yadda yadda yadda.
An AFB stains red, but it is a different procedure than a gram stain so it has nothing to do with that red. AFB have gram positive cell walls so they stain red (and not purple) because it’s a different procedure. You wouldn’t do a gram stain on a AFB (like if you suspect someone has a tuberculosis) because if you did the gram stain the bacteria would be gram positive and wouldn’t tell you it’s tuberculosis (or a AFB). The acid fast stain provides a more definitive diagnosis since it targets all mycobacteria. The presence of one single AFB in sputum or tissue confirms a diagnosis in most cases of tuberculosis.
