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Flashcards in Bacteriological Stains Deck (16)
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List, in order, the reagents used in the Gram stain and tell the purpose of each step.

Reagent: Crystal violet, Gram's iodine, Alcohol, acetone, or combination, Safranin
Function: Primary stain, Mordant, Decolorizer, Counterstain
Action: Stains all cells purple, Fixes crystal violet to cells, Removes crystal violet from gram-negatives, Stains gram-negatives pink


What accounts for the different staining characteristics of gram-positive and gram-negative cells?

The different cell wall compositions. Teichoic acid cross-links in the thick peptidoglycan layer of the gram-positive cell wall enable it to resist decolorization. Large amount of lipopolysaccharide in the gram-negative cell wall allows decolorizer to enter.


A technician performs a Gram stain of a colony growing on a blood agar plate. After careful examination, no bacteria can be located on the slide. What error might have been made?

The technician might have forgotten to heat fix the slide. Without fixation the specimen may be washed off during the staining process.


What quality control is required for the Gram stain?

A slide is prepared using gram-positive organisms such as S. aureus and gram-negative organisms such as E. coli. The gram-positive organisms should appear purple and the gram-negative organisms pink. Gram stains should be checked for each new batch and at least weekly.


A direct smear of a specimen reveals many gram-negative cocci in chains. A control slide run at the same time reveals gram-negative cocci and gram-negative bacilli. What should be done?

Since the Gram stain control slide does not give the expected results (gram-positive cocci and gram-negative bacilli), the procedure should be repeated. The slides were probably overdecolorized. The decolorizer should be added drop by drop just until it begins to run clear.


What is the term to describe bacterial cells of one species that vary in size and shape?



Name two things that might cause gram-positive bacteria to stain gram-negative.

A culture that is too old or overdecolorization during the staining process.


All of the polys on a Gram stain appear blue. What does this mean?

The slide was underdecolorized.


What is the significance of many polys on the Gram stain of a stool?

It indicates an infection due to an invasive organism such as enteroinvasive E. coli, Salmonella, Shigella, Campylobacter, or CLostridium difficile.


A colony growing on a chocolate agar is Gram stained. The organisms stain pink and vary in size and shape. Some appear as a very short rods, while others are longer. How should these findings be reported?

Pleomorphic gram-negative rods.


Which enteric pathogen is associated with RBCs on a Gram stain of the stool?

Enterohemorrhagic E. coli (EHEC). Serotype O125:H7 is most common.


How do you yeast stain on a Gram stain?



What Gram stain findings are indicative of bacteriuria?

One or more bacteria per oil immersion filed on a uncentrifuged urine. This correlates to a colony count of 10^5 CFU/mL.


How can the Gram stain be used to assess the acceptability of a sputum specimen?

It helps to differentiate sputum from saliva. A good sputum will have 25 or more polys and loss than 10 epithelial cells per low field (unless the patient is neutropenic). Saliva will have more than 25 epithelial cells per low power field.


How is a Gram stain of CSF prepared?

From the sediment. The CSF should be centrifuged for at least 15 minutes at 1500X g. Many labs use a cytocentrifuge preparation for increased sensitivity.


How is the acridine orange stain used in microbiology?

Acridine orange is a fluorochrome dye that stains both gram-positive and gram-negative bacteria, living or dead. It is used to located bacteria in blood cultures and other specimens where they are difficult to see with the Gram stain.