| Question | Answer |
| What are the general characteristic of Mycobacteria? | -obligate anaerobes 5-10% CO2
-slow growing 2-6 weeks
-non spore-forming, non motile
-slender, slightly curved or straight rods
*high lipid content on cell wall so they are acid fast |
| How are Mycobacteria identified? | based on growth rate, colony morphology, incubation temperature, and biochemical reaction |
| How should expectorated sputum be collected and handled? | Spontaneous expectorated sputum
• 5-10 ml of specimen with little oral flora
• Best done 3-5 consecutive days
• Early morning specimen |
| How should aerosol induced sputum be collected and handled? | Aerosol induced sputum
• NaCl mist-nebulizer. This is done by respiratory therapy
• Early morning specimen is best
• Advantages:
1. Fewer contaminates
2. Faster growing |
| How should gastric lavage be collected and handled? | 1. Done 1/2 hr. after aerosol-induced sputum - collected to
obtain swallowed sputum
2. Overnight fast
3. gastric + induced sputum combo yield more positive cultures than alone
4. Disadvantage: frequently contaminated
with commensal Mycobacteria |
| How should urine be collected and handled? | Urine
1. Early morning clean catch
2. 3-5 specimens
3. 24 hour collection is not recommended |
| How should stool be collected? | Stool
1. Naturally passed
2. 3-5 consecutive days |
| How should Blood be collected? | 1. Sterile technique for draw
2. Special blood culture bottles (BACTEC or BACT/Alert) –
continuous monitoring
3. Isolator lysis-centrifugation system
4. Sodium heparin tubes collected and sent to Mayo within 48
hours |
| How should tissue specimen be collected and handled? | Tissue Specimen
1. Grind
2. Incubate cutaneous tissue cultures at 35oC and 30oC |
| How should sterile body fluid be collected and handled? | 1. Sterile container or capped syringe, preferred
2. Anticoagulant could be added to fluids that may clot (citrate or heparin)
3. Swab is Discouraged, unless E-swab |
| What is Digestion of non-sterile specimens? | Digestion:
• breaks up mucous and cells in specimen to liberate A.F.B. |
| What is the purpose of timed decontamination process of non-sterile specimens? | • Kills normal flora and contaminating bacteria without
harming A.F.B.
• Use strong acidic or alkaline solutions - the lipid content of
A.F.B. cell walls protects them from extremes of pH
• Decontamination increases the chances of recovering
A.F.B. |
| What is the procedure of the traditional method of Digesting and decontaminating specimen? | NaOH 4% is the "traditional“ method to digest and decontaminate • Time carefully; needs to stand for 15 minutes
• Neutralize sediments (phenol red indicator • 5% Oxalic acid - useful to decontaminate specimen contaminated with Pseudomonas (CF patients) |
| What is the procedure of the gold standard method of Digesting and decontaminating specimen? | NALC-NaOH 2% "gold standard“
• N-acetyl-L-cysteine is the mucolytic agent
• NaOH 2% is the decontaminant
• Neutralize with phosphate buffer
• If after using the NALC method a specimen is still viscous
a small amount of crystalline NACL can be added |
| What are properties of A.F.B (acid fast bacteria)? | • Cell walls have high lipid content enabling A.F.B. to retain
stain when decolorized with acid alcohol
• Stain morphology - slender, curved, pointed and beaded |
| What is the procedure to stain AFB using carbolfuchsin | 1. Carbolfuchsin - Fuchsin with phenol a) Primary stain: Carbolfuchsin b) Decolorizer: acid alcohol c) Counter stain: methylene blue d) Two techniques: 1. Ziehl-Nielsen "hot stain“
2. Kinyoun "cold stain“ e) A.F.B. stain red; other bacteria stain blue |
| What is the procedure to stain AFB using fluorochrome dye? | Fluorochrome dye
a) Primary stains:
1. Auramine
2. Rhodamine
b) Decolorizer: acid alcohol
c) Counter stain: potassium permanganate
d) Screen slides using fluorescent scope on 25 or 40 objective
e) A.F.B. will stain yellow against black background |
| What are the advantages of using fluorochrome dye? | 1. More fields scanned in less time
2. Yield higher number of positive stains
3. Questionable smears can be overlaid using Carbolfuchsin
method |
| What is the disadvantage of using fluorochrome dye? | Dead mycobacteria will stain with fluorochrome method |
| Why are Stain results important? | 1. Early detection means early treatment
2. Monitor response to drug therapy
3. Must be paired with culture
• 10,000 AFB/ml = positive smear
• Sensitivity of 22-80% |
| What are Non-selective mycobacteria media? | 1. Egg based media
2.Agar based media |
| What are broth media used for mycobacteria? | 1.Middlebrook 7H9
2.Blood culture systems to detect A.F.
• BACTEC systems
• BacT/Alert system
• Septi-Chek bottles
• Isolator Lysis-Centrifugation system
3. MGIT 960 system: Modified 7H9,
non-radiometric microbial detection system |
| What is used to interpret cultures? | 1. Testing of bottles -Culture held six weeks -
Bottles are continuously monitored and
automatically flagged as positive when growth
is detected
2. Primary L-J slants- checked weekly |
| Preliminary identification made based on what? | Colony morphology: smooth or rough
▪ Rate of growth: rapid grower (colonies in <5 days)
▪ Optimal temperature of growth
▪ Pigment production: buff vs. yellow
▪ Photoreactivity |
| Name some mycobacteria and their optimal growth temperatures | - most grow at 35°
- 42°-M. xenopi,
- 30-32°-M. marinum, M. ulcerans, M. haemophilum |
| What are the different type of photo reactivity? | ▪ Photochromogens: produce pigment when exposed to light
▪ Scotochromogens: produce pigment in light or dark
▪ Non-chromogens: No pigment formation |
| How is Niacin accumulation tested? (media used & interpretation) | ■ an LJ slant with >50 colonies that are 3 weeks old need to
be tested for niacin.
■ If niacin is present a yellow color will develop. |
| What is the Nitrate reduction test? | Test if bacteria conduct nitrate reduction, if they have the gene for the nitrate reductase enzyme. Red is positive for nitrite.
1: nitrate reduction to nitrite
2: nitrate reduction to ammonia
3: no reduction of nitrate |
