1. Intro (I) Flashcards
Bacteria that are not stained through Gram Staining
Chlamydia
Legionella
Mycoplasma
Gram stain procedure
Crystal violet (1° stain)
Gram’s iodine (Mordant)
Acetone / alcohol (Decolorizer)
Safranin (Counterstain)
COAM
What is the difference between Gram+ and Gram- cell walls?
Cell walls of gram-negative cells have higher lipid content that gram-positive cells
Special consideration of Gram Stain of sputum specimen; if >25 epithelial cells/lpf
Saliva
Special consideration of Gram Stain of sputum specimen; few epithelial cells, many PMN cells
Specimen more likely to yield a pathogen
When can you say that a sputum specimen is actually saliva?
When there are >25 epithelial cells/lpf
When can you say that a sputum specimen may likely yield a pathogen?
When there are few epithelial cells and many PMN cells
Special consideration for urine specimen
1 cell per oil immersion field = approx. 1x10^5 CFU/mL
Enumerate the quantitation criteria for Gram Stain
- no organisms seen
- Few per slide = rare
- 0 to 2 per field = few
- 2 to 10 per field = moderate
- more than 10 per field = many
Constituents of media
Agar Nutrients Enrichments Buffers pH indicators Inhibitors
BEANpI
Constituent of media; gelatinous seaweed extract
Agar
Percent of agar in plates
1-2% agar in plates
Nutrients in media can be _______ or _______
Hydrolyzed proteins (animal or plant) Carbohydrates/sugars
Enrichments used in media
Yeast extracts
Blood
Purpose of buffers in media
Provides a stable pH for growth
pH indicators used in media and their colors
Neutral red: red to colorless
Phenol red: yellow to red
Thymol blue: yellow to green/blue
Inhibitors in media
Bile salts Antibiotics Dyes Sodium chloride Sodium citrate
BADSS
Examples of dyes used as inhibitors in media
Crystal violet
Eosin
Methylene blue
Example of bile salt used as inhibitor in media
Sodium deoxycholate
Agar that contains inhibitory agents to all organisms except one being sought
Selective agar
Example of selective agars
Colistine nalidixic acid agar (CNA)
MacConkey Agar
Agar that selects for certain organisms to the disadvantage of others
Selective agar
Agar that allows organism to be morphologically distinguished from other organisms with different characteristics
Differential agar
Examples of differential agar
Sheep blood agar (SBA)
MacConkey agar
Supportive media aerobic cultures
Blood agar
Chocolate agar
Selective/differential media for gram-negative aerobic bacilli
MacConkey agar
Xylose Lysine Desoxycholate (XLD) agar
Hektoen Enteric Agar (HEA)
Selective/differential media for gram-positive aerobic organisms
Colistin Nalidixic Acid (CNA) agar
Phenylethyl Alcohol Agar (PEA)
Purpose of Blood Agar for aerobes
General growth of gram-positive and gram-negative aerobes
Why is the Blood Agar also considered as differential aside from being supportive?
Because organisms may demonstrate hemolytic patterns
Chocolate agar is used for
Isolation of fastidious organisms:
- Haemophilus spp.
- Neisseria spp.
- Brucella spp.
- Capnocytophaga spp.
Components of MacConkey agar
Lactose
Crystal violet
Bile salts
pH indicator in MacConkey agar
Neutral red
Purpose of lactose in MacConkey agar
- Provides fermentable sugar (Lactose positive vs negative)
- Lactose fermenting vs non-lactose fermenting organism
Purpose of crystal violet in MacConkey agar
Inhibits gram-positive bacteria
Purpose of bile salts in MacConkey agar
Inhibits gram-positive bacteria
MacConkey agar is selective for
Gram-negative organisms
Lactose positive bacteria (lactose fermenters)
Escherichia coli: dry, flat, dark pigment Enterobacter: mucoid Citrobacter: late fermenter Klebsiella: mucoid Serratia: late, red pigment (some)
EECKS
Lactose negative bacteria (non-lactose fermenters)
Proteus: swarming Providencia Pseudomonas Hafnia Edwardsiella Morganella Salmonella Shigella
P3HEMS2
Component of XLD; inhibits gram-positive organisms
Sodium deoxycholate
pH indicator of XLD
Phenol red
Carbohydrate components in XLD
Lactose
Sucrose
Xylose
Carbohydrates in XLD that provide fermentable sugar
Lactose
Sucrose
Enzyme detected by lysine in XLD agar
Lysine decarboxylase
Effect of Salmonella to lysine in XLD agar
Since Salmonella has lysine decarboxylase, the lysine in the agar is decarboxylated, shifting the pH indicator to red
Component of XLD; detects production of H2S
Sodium thiosulfate
Ferric ammonium citrate
Characteristics of E. coli in XLD
Yellow color; ferments carbohydrates; causes large pH drop
Characteristics of Shigella and Providencia in XLD
Colorless or red; no fermentation; no H2S
Characteristics of Salmonella in XLD
Red with black center; ferments xylose; produces low pH; then decarboxylates lysine; produces high pH; H2S production
Purpose of sodium deoxycholate in XLD
Inhibits gram-positive organisms
Organism(s) that ferments carbohydrates in XLD, causes large pH drop, yellow color
E. coli
Organism(s) that is/are colorless or red in XLD; no fermentation, no H2S production
Shigella
Providencia
Organism that produce red color with black center in XLD, ferments xylose, produces low pH, then decarboxylates lysine, produces high pH, H2S production
Salmonella
What is the X factor in the chocolate agar?
Hemin
IsoVitaleX Enrichment (according to Elsevier reviewer)
What is the V factor in the chocolate agar?
Nicotinamide adenine dinucleotide (NAD)
Hemoglobin (according to Elsevier reviewer)
Developer of Gram Stain
Hans Christian Gram (1884)
