mycobacteria???!!!! Flashcards
mycobacteria gram stain poorly why? what is the consequence of this on a gram stain?
the mycolic acid / lipids in their cell walls don’t hold/pick up the crystal violet / and safranin
- ghost cells: faint unstained images
- gram neutral
what should you do with a mycobacteria specimen if it’s not processed asap? What type of specimen is the exception to this rule?
refrigerate the specimen, unless it’s a blood one
what are three names given to mycobacteria that are not M leprae or M. tuberculosis?
nontuberculous mycobacteria (NTM), mycobacteria other than tuberculosis (MOTT), or atypical mycobacteria
NTM is commonly found in _______ and _____. They are/aren’t considered transmissible from person to person
water and soil
AREN’T transmissible from person to person
how is primary tuberculosis transmitted, and where does it go to in the body? What can latent organisms lead to?
spread via droplet nuclei
latent organisms can reemerge and cause
- secondary/reactivation tuberculosis
what does M. tuberculosis cause that are tumor like, inflammatory lesions? What else are these called?
MTB cells are sometimes called ______? What do they look like?
cause granulomas, also called tubercles
MTB cells are sometimes called tubercle bacilli: necrotic centers with a soft, cheesy appearance. caseous
active tuberculosis can cause what disease of the bones? What are symptoms of TB?
TB bone disease = Pott’s disease
symptoms of TB: coughing, hemoptysis (coughing up blood), weight loss, and low-grade fever
what is used for the tuberculin skin test? What do positive results look like?
Purified protein derivative (PPD), a MTB antigen that MTB people have antibodies to
pos: red, hard area at the injection site within 48 - 72 hrs
pos results do not distinguish between latent and active infections
M. avium complex (M. avium and M. introcellulare) is associated with what disease?
M. avium complex is the most common cause of ______ infections and is resistant to many __________ ______
associated with AIDS (GI or disseminated disease)
MAC most common cause of NTM infections and resistant to antimicrobial drugs
the M. fortuitum complex includes what three species of mycobacteria? What kind of infections are these common to see in?
M. fortuitum, M chelonae, and M. abscessus
seen in wound infections
M. haemophilum causes what three things, and needs what three things to grow?
causes
- skin ulcers
- lymphadenitis
- disseminated disease
needs:
- hemin
- hemoglobin
- ferric ammonium citrate
only grows on chocolate agar
M. kansasii causes NTM pulmonary disease in what areas? What three biochemical tests is it positive for?
Southern/western states (eg Texas, Louisiana, Florida, Missouri, California)
- tween 80, nitrate, and SQ catalase pos
second leading cause of NTM lung disease, (first is M. avium complex)
M. leprae causes ________, also known as __________ disease, a disease of the skin, mucus membranes, and peripheral nerves. What animals is it found in?
leprosy; Hansen’s disease; foot pads of mice and armadillos
M. marinum causes _________-______ granulomas. Likes to grow in ____ or _______ water
swimming-pool granulomas
grows in salt or fresh water
M. scrofulaceum causes what?
cervical lymphadenitis
what mycobacteria is the “tap-water bacillus/contaminants”?
M. gordonae
what are the parameters of respiratory tract specimens?
sputum/bronch washes:
- 3 - 5 cultures on different days
- 5-10 mL
- collected in early morning
- 4 hr window after collection unless sodium bicarbonate is added
sputum must be decontaminated
what are inappropriate AFB specimens?
swabs, 24 hr pooled sputum (are more contaminated than non-pooled specimens), 24 hr pooled urine specimens
what are the three specimen processing steps for AFBs, and what is done during each?
concentration: centrifugation for 15 minutes at 1500 RPMs
decontamination: mycobacteria resist alkaline or acidic chemical agents that kill of normal bacteria due to their high lipid content
- contamination rate of 2 - 5% recommended
digestion: frees the mycobacteria from clumps of protein and allows mycobacteria to sediment during centrifugation
What are digestion reagents?
NALC + 2% NaOH used within 24 hrs of preparation
- NALC = mucolytic agent
- NaOH = decontaminates specimen
phosphate buffer added: lowers sg of specimens to enhance sedimentation
albumin = helps specimen sediments adhere to solid culture media/microscope slides
digestions steps
10 mL specimen and 10 mL of NALC-NAOH in conical and vortexed. Left at RT for 15 mins. for the NaOH to decontaminate. Add phosphate buffer, centrifuge for 15 min at 3000g and supernatant decanted. Albumin added to sediment. Acid fast stain.
What must be used in conjunction with acid fast smears, and why? How many organisms per milliliter in a culture must be present to be considered positive?
cultures must be used in conjunction to acid fast smears because even though they take longer to show AFB growth, they are more sensitive than smears
needs 5000 - 50000 organisms per milliliter in culture to be pos
what are the two types of acid fast stains, and what are the two staining procedures for each?
- carbolfuchsin (contain phenol and fuchsin)
- Ziehl-Neelson (hot method): pos organisms are red, neg are blue
- Kinyoun (cold method) - fluorochrome
- auramine O / auramine-rhodamine: has phenol to penetrate AFB walls
auramine-rhodamine stain must sit for ___ minutes and then decolorized with ____ for 2 minutes. Acid fast organisms are ______ color. The slide is counterstained with _______ or ________
15 minutes
decolorized with acid-alcohol for 2 minutes
AFBs are yellow - yellow-orange
counterstain with potassium permanganate or acridine orange
Carbolfuchsin-stained smears should be looked at how many fields / how many passes? What about for Fluorochrome stained smears?
C smears: 300 fields, 3 long passes and 9 short passes
F stain: 30 fields, 3 long passes
what are five egg-based media, and what do they all have that inhibit contaminating organisms?
nonselective:
1. petragnami (very high conc. of malachite green)
2. ATS (low malachite green)
3. Lowenstein-Jensen medium: most commonly used egg medium
selective:
1. LJ-Gruft
2. mycobactosel-LJ
all have malachite green
agar based media for AFBs have a variety of nutrients and little malachite green. What are the selective and nonselective types?
nonselective:
1. middlebrook 7H10 and 7H11
selective:
1. mitchison’s 7H11
2. Mycobactosel-middlebrook 7H11
liquid media for ABFs contain what? What does this do? What are the kinds of liquid media?
has tween 80: surfactant that breaks up clumps of AFB so the organisms can disperse throughout media. Mycobacteria grow faster in liquid media
types:
- dubos tween albumin broth
- middlebrook 7H9, 7H12, 7H13
most mycobacteria grow at what temps? Which ones have a lower optimal temp? What do we do when we suspect these organisms?
most grow at 35 - 37º C
lower temp: M. marinum, M. haemophilium, M. fortuitum complex, and M. ulcerans
two sets of media:
- one at normal AFB temp
- one at 24 - 35º C for the AFBs above minus M. fortuitum
middlebrook media must be incubated in ____
CO2
what are the two BACTEC mycobacteria systems, and what do each do?
460 TB system: radioactive carbon, 14CO2 formed if mycobacteria is present
9000 MB system: detection of O2 consumption as mycobacteria grow –> fluorescence present if mycobacteria is present
how does the MB/BacT system detect mycobacteria?
CO2 sensor in a broth medium that turns yellow from original green color if mycobacteria is present
what are the three kinds of chromogens (photoreactivity test where AFBs produce carotenoid pigments)
photochromogens: produce yellow to orange pigments in the light only
scotochromogens: make deep yellow to orange pigments in the light and dark
nonphotochromogens: make no pigment at all. (look buff, tan, pale yellow colonies)
nearly all mycobacteria are ________ positive, but what are the three tests that can help differentiate the kinds of mycobacteria?
catalase pos
- heat stable 68ºC catalase: some mycobacteria can be heated to 68ºC while other cat pos ABFs will be inactivated
- uses Tween 80 H2O2
- bubbles in heated or non heated tube is pos result - semi-quantitative catalase:
- tween 80 H2O2 added to LJ slant and height of bubbles made is measured (> 45 mm = high catalase producer) - drop method:
- some isoniazid resistant MTB are cat neg, where no bubble means INH resistance
iron uptake: some mycobacteria can convert _______ _________ _____ to ______ _____. What do pos results look like?
some mycobacteria can convert ferric ammonium citrate to iron oxide
pos results: rusty to red-brown color on a tan medium
all mycobacteria can produce _________ (nicotinic acid), but only some can turn it into _______ (precursor to NAD).
Steps: grow AFB on LJ slant for many weeks, add water, wait ____ minutes, add ______ ______ and ________. What color is a pos result?
niacin to nicotinic acid mononucleotide
Steps: grow AFB on LJ slant for many weeks, add water, wait 15 minutes, add cyanogen bromide and aniline.
Yellow pos result
Nitrite reduction test reagents are what three things (that are added to mycobacterial isolate with nitrite substrate for 2 hrs)?
- dilute HCl to acidify the medium
- sulfanilamide
- N-naphthylethlenediamine hydrochloride
add zinc if red color doesn’t appear
pyrazinamidase (PZA) is an enzyme that deaminates pyrazinamide to form what two things? How is a pos pink color made?
pyrazinamide to
1. pyrazinoic acid
2. ammonia
the pyrazinoic acid with ferrous ammonium sulfate makes a pink color
tellurite reduction of tellurite to tellurium pos and neg colors
white = neg
black = pos
what are the MTB complex AFBs?
- M. tuberculosis
- M. bovis
- M. africanum
what are the two photochromogens?
M kansasii
M. marinum
what are the three scotochromogens?
M. gordonae
M. scrofulaceum
some M. xenopi
what are the four nonphotochromogens?
M. avium complex
M. haemophilum
M. ulcerans
most M. xenopi
what are the fast grower AFBs?
M. fortuitum complex (all nitrate and iron pos)
- M. fortuitum
- M. chelonae
- M. abscessus
what are the two first line anti-TB drugs used to treat TB?
isoniazid (INH) and rifampin (RIF)
