BACTE Flashcards
best time to collect any type of spx for bacteriology
Acute phase of illness (w/n 72 hrs)
T/F
It is acceptable to collect spx once antibiotic therapy has started
F
no/less organism recovered
technique STRICTLY observed during collection and processing
Aseptic technique
allowable duration of the transportation of collected samples (*w/o delay)
30 mins – 2 hrs
LEVEL OF PRIORITIZATION
- Critical / Invasive
- UNPRESERVED
- Quantitation required
- Preserved
- Batch processing
example of critical spx or obtained thru invasive procedures
CSF
amniotic fluid
blood
pericardial fluid
heart valves
example of unpreserved spx
feces
sputum
wound drainage
example of spx with quanti required
catheter tip
urine
tissues for quantitation
ex of preserved spx
urine, feces, swab in holding media
ex of spx for batch processing
sputum
AFB culture
performed using colony counting to r/o UTI; delay w/ quantitation may affect the count
Urine C/S
PURPOSE OF BLOOD CULTURE
r/o:
Bacteremia
Septicemia/sepsis
high no. of bacteria in the blood but NOT ENOUGH TO CAUSE HARM or S/S to px
Bacteremia
high no. of bacteria in the blood, causing harm (S/S) to the px
Septicemia/sepsis
no.1 cause of community-acquired UTI; GIT flora
E. coli
Ex of Blood pathogens
E. coli
P. aeruginosa
S. aureus
blood pathogen that is NEVER a normal floraa
P. aeruginosa
most common cause of sepsis
S. aureus
normal flora but affects anterior nares
S. aureus
Process of cleansing the phleb site prior to collection to ensure that the blood submitted for culture is not contaminated
70-95% alcohol –>
Iodine scrub –>
Alcohol rinse
Common contaminants in blood submitted for culture
Viridans streptococci (S. anginosus, S. salivarius, S. sanguinis, S. mitis, S. mutans)
Staphylococcus epidermidis
Propionibacterium acnes (recent: Cutibacterium acnes)
Dilution of Blood to Media
1:10
Duration of Routine blood culture
7 days
Duration of Leptospirosis detection
8 weeks
Gold std for Leptospirosis detection?
Spx required?
MAT – Macro/micro agglutination test
SERUM – Ab detection
used in micro agglutination test
live leptospira
used in macro agglutination test
heat-killed leptospira
Duration of Brucellosis detection
3-4 weeks
Rapid test for Brucellosis detection
Optimal spx?
SAT (serum agglutination test)
Blood
T/F
All brucella spp. are animal normal flora but not of man.
T
In man: causes febrile disease (recurrent fever)
4 signif. brucella spp.
abortus
suis
canis
melitensis
T/F
brucella are culturable but culture is not usually done
T
most preferred test - SAT
no. of sets to be collected for blood culture
2-3 (usually 2 in practice)
best time to collect blood for blood culture
During peak of fever
blood amount to be collected for blood culture in the ff:
adult
pediatric
infants
Adult: >20 mL
Pediatric patients: 1-10 mL
Infants: 0.5-1 mL
How is 2 time collection done for blood culture?
both arms; 1 hr interval (2 sets: for aerobic & anaerobic culture)
1-time collection is only allowed in these cases. How many mL of blood must be collected for blood culture?
STAT cases
40 mL
routine anticoagulant for bacterial blood culture
0.025% SPS
disadvantage of 0.025% SPS
what is the remedy
inhibit: Neisseria, Gardnerella
+1% gelatin
anticoagulants used for molecular procedures only (PCR)
White top tube w/ EDTA
Media used for Blood culture
- Tryptic Soy Broth (TSB)
- Brain Heart Infusion (BHI) agar
- Brucella broth – esp. for brucellosis det.
- Castaneda bottle - brucella det. (biphasic)
spx collected to r/o bacteremia and septicemia
blood
spx collected to r/o bacterial meningitis
CSF
Process of CSF collection
Lumbar tap (invasive)
CSF tube no. used for micro
2
*if only 1 submitted, micro is the priority
Storage temp of CSF
37C
Transport temp of CSF
RT
CSF processing
Centrifuge CSF →
remove supernatant →
left are sediments (for smear preparation & culture use)
USUAL BACTERIAL ISOLATES IN CSF
Streptococcus agalactiae
Haemophilus influenzae serotype b
Streptococcus pneumoniae
Neisseria meningitidis
Listeria monocytogenes
agent of neonatal meningitis isolated in CSF
Streptococcus agalactiae
agent of children (<5 y/o) meningitis isolated in CSF
Haemophilus influenzae serotype b
agent of adult (>29 y/o) meningitis isolated in CSF
Streptococcus pneumoniae
agent of meningitis in 5-29 y/o isolated in CSF
Neisseria meningitidis
agent of meningitis in elderly/immunocompromised isolated in CSF
Listeria monocytogenes
To do after CSF collection from a shunt
Add thioglycolate
Very versatile media; allows growth of facultative anaerobes, strict aerobes, anaerobes
thioglycolate
spx to r/o repeated UTI
urine
usual request to r/o repeated UTI
Urine culture & sensitivity (C/S)
specimen of choice for bacterial culture to r/o repeated UTI
Clean catch midstream urine
spx for those unable to void urine
Catheterized urine
urine spx for anaerobic culture
Suprapubic urine
urine spx for molecular studies like PCR
First morning urine
CAUSATIVE AGENTS OF UTI
E. coli
S. saprophyticus
Klebsiella
Enterococcus faecalis
no. 1 cause of community-acquired UTI
gram negative rod; normal GIT flora
E. coli
1 cause of UTI in young female and in elderly
women with catheter
S. saprophyticus
same family w/ E. coli; under Enterobacteriaceae that can also cause UTI
Klebsiella
gram + cocci that can cause UTI
Enterococcus faecalis
Determines the # of colonies / mL of urine by using a colony counter (ideal) or by manual counting
Colony count
loop required in colony count
calibrated loop (1 or 10 uL)
*determine DF
DF if loop size is 1 uL
1000
DF if loop size is 10 uL
100
formula for colony count
of colonies counted x dilution factor = colony count/ml of urine
colony count considered as UTI
> 100,000
media used in colony count
MHA
*usual media for susceptibility testing
preferred urine preservative; maintains accurate colony count
Boric acid
spx to r/o LRTIs (pneumonia, bronchitis, TB)
sputum
Patient prep for sputum collection submitted for bacterial culture
Patient must be instructed to rinse/gargle with water prior to collection and cough deeply into container (5-10 mL sputum)
amt. of sputum required for bacterial culture
5-10 mL sputum
what must be done when spx submitted for sputum culture is NONSTERILE
evaluate quality → Bartlett’s Classification
checks if the spx is sputum or saliva? what is the criteria to be considered as SPUTUM (TRUE RESPIRATORY SAMPLE)?
Bartlett’s Classification
SPUTUM (true respiratory sample) - alveolar macrophage
SALIVA (not a respiratory sample) - >10 SECs, <25 PMNs
CAUSATIVE AGENTS OF PNEUMONIA
Pseudomonas aeruginosa
Klebsiella pneumoniae
Streptococcus pneumoniae
Haemophilus influenzae
cause pneumonia in those w/ cystic fibrosis
Pseudomonas aeruginosa
1 cause of ventilator-associated pneumonia (intubated px)
Pseudomonas aeruginosa
TB agent; slow-growers (take weeks to produce growth)
Mycobacterium tuberculosis
M. tuberculosis is what type of BSL pathogen? culture of this organism must be done using BSC class?
BSL 3 pathogen
BSC Class 2
sputum for TB identification must undergo:
Decontamination – remove contaminants and normal flora
Digestion – liquefy the viscous sputum to free any trapped organisms
Gold standard for digestion & decontamination
N-acetyl-L-cysteine (NALC) + NaOH
NALC – mucolytic/digestant
NaOH – decontaminant
Frequently used agent; readily available; both a decontaminant and digestant
4% NaOH
T/F
4% NaOH only act as decontaminant when + w/ NALC
T
Added to sputum likely to contain P. aeruginosa
5% oxalic acid
T/F
All spx for TB det. requires decontamination & digestion
F
only non-sterile spx
types of sputum collection and no. to be considered (+)
1 spx collected in 3 days
(+): 2 out of 3
2 spx in 1 day
(+): 1 out of 2
Recommended sputum collection by DOH (to reduce TB cases)? Type of sputum spx needed in each collection?
2 specimens in 1 day
(+): 1/2
1st collection – first morning sputum
2nd collection – random sputum
Throat swab/Nasopharyngeal swab (NPS) is the spx for ID of?
Streptococcal infections (ex. pharyngitis) - S. pyogenes
Corynebacterium diphtheriae
Viridans strep
major throat pathogen
Streptococcus pyogenes
major throat flora
Viridans strep
major cause of subacute bacterial endocarditis (SBE)
Viridans strep
implicated in dental carries due to production of glucans and dextrans (enhances teeth attachment)
Viridans strep
spx collected to detect Bordetella pertussis
NPS
causes whooping cough
Bordetella pertussis
specimen processed to detect carrier state of N. meningitidis
NPS
spx required if N. meningitidis found in naso & oropharynx is normal flora (non-encapsulated)?
NPS
spx required if N. meningitidis is found to be pathogenic (encapsulated)?
CSF
Neisseria spp. is sensitive to 2Cs
cold temperature
cotton swab - contain fatty acids
Alternative: SWAB MADE FROM DACRON or CALCIUM ALGINATE
Swabs are only suitable for what type of bacteria
AEROBIC
Specimen to collect for culture when patient is on a ventilator/ intubated
Endotracheal aspirate
Not to be used for VIRUSES; inhibits replication
Calcium alginate
spx to r/o food poisoning, diarrhea, gastroenteritis
stool
GIT pathogens
Salmonella
Shigella
(never a normal flora of GIT)
invasive bacteria that causes bloody diarrhea
non-lactose fermenters & nonmotile (in general)
Shigella
4 Shigella serogroups
A – S. dysenteriae
B – S. flexneri
C – S. boydii
D – S. sonnei (LLF)
GIT pathogen with only 2 spp.? What are those?
Salmonella
S. enterica – human pathogen
S. bongori – animal pathogen
may be collected if stool collection is not possible
Rectal swab
Once stool is received, it is streaked immediately in?
EMB
MAC
SSA
manner of stool streaking for culture with 4 quadrants streaked on plated media
Semi-quantitation technique/isolation streak
what is isolated in stool streaked using semi-quanti technique?
area 4 - with isolated single colonies
automated program by ID of species through series of biochem tests and perform susceptibility testing
strictly requires PURE ISOLATES
VITEK system
Examples of Critical Values in Microbiology
- Positive blood culture
- Positive CSF gram stain or culture
- Positive cryptococcal antigen test or culture
- Positive blood smear for malaria
- S. pyogenes from a sterile site
- Positive acid-fast smears or positive Mycobacterium culture
- S. agalactiae or HSV from genital site of a pregnant woman at term
- Detection of significant pathogen (ie. B. pertussis, Brucella, Legionella)
What to do when critical values/ panic values are noted in micro:
REPORT THE RESULT ASAP
Types of bacteria processed in the lab:
Facultative anaerobes
Aerobes
Strict aerobes
bacteria that must not be exposed to air during incubation? what is required to prevent air exposure?
Facultative anaerobes
Gaspak Jar
bacteria requiring 21% O2 + 0.03% CO2 (placed at 37ºC, 18-24 hrs)
Aerobes
other aerobes requiring ↑ CO2
CAPNOPHILIC
(must be ensured that they are supplied with inc. CO2 by using a candle jar)
gaspak jar component that maintain anaerobiasis
Palladium pellets /catalyst
function of indicators in gaspak jar? give an example and its corresponding negative and positive color
ensure no presence of air
In O2 presence:
Methylene blue (BLUE)
Resazurin (PINK)
In O2 absence:
Colorless
Common failure of GasPak
inactivation of catalyst due to repeated use
CO2 needed by capnophilic bacteria? give an example of this bacteria
5-10% CO2
Neisseria
bacteria that is both Microaerophilic (5% O2) & Capnophilic
genus Campylobacter
Darting motility
Cause gastroenteritis
Stool pathogens
Microscopic appearance: wings of seagulls
genus Campylobacter
Anaerobic culture (gaspak jar) contents
5% CO2
10% H2
85% N2
Microaerophilic culture (candle jar) contents
5% O2
10% CO2
85% N2
2 general AST methods
Dilution method (quantitative)
Disk diffusion method – Kirby Bauer (qualitative)
Lowest concentration of the antibiotic that inhibited bacterial growth
Minimum Inhibitory
Conc. (MIC)
Lowest concentration of antibiotic that can kill bacteria
Minimum bactericidal conc./ Minimum lethal conc. (MBC/MLC)
Required depth of MHA for Kirby Bauer
3-5 mm
swab used and manner of streaking in MHA during Kirby Bauer tech
sterile cotton swab
overlap streaking
Size of antibiotic disk used in Kirby Bauer Technique
6mm
distance of the disks from the center used in Kirby Bauer Technique
24 mm
distance between 2 disks used in Kirby Bauer Technique
15 mm
RULE of stacking of plates in incubator for AST (kirby bauer)
not be >5 plates in 1 column
Incubation temp and duration of plates for Kirby Bauer
37°C for 16-18 hours
result in ZOI when:
- too thin agar
- very dry agar surface
- too light inoculum
Larger ZOI
False sensitive
result in ZOI when:
- too thick agar
- too much moisture on agar surface
- too heavy inoculum and too long incubation time
Smaller ZOI
False resistant
allowable no. of antibiotic disks on a 150 mm plate
No >12 disk
allowable no. of disks on a 100 mm plate
No >5 disk
Storage temp for antibiotic disks-working supply
2-8°C
Cell wall inhibitors
Beta lactams: Penicillin, Cephalosporins, Carbapenems
Glycopeptide: Vancomycin
Protein synthesis
inhibitors
Aminoglycosides: Gentamycin
Tetracycline
MLS (macrolide-lincosamide-streptogramin) – Erythromycin, Clarithromycin
AST of Streptococci is done using what media
Use MHA + blood (required since strep is fastidious)
Detects inducible clindamycin resistance among strains of S. aureus
D-test (double disk diffusion test)
Required when initial result of Erythromycin to Clindamycin is DISCREPANT
D-test (double disk diffusion test)
D-test (double disk diffusion test) is used to confirm the true result of what antibiotic
CLINDAMYCIN
used in D-test
15 ug Erythromycin
2 ug Clindamycin
*15 mm apart
gene that activates clindamycin resistance
erm gene
POSITIVE result of D-test? What does it indicate?
blunting/flattening of Clindamycin zone to produce D pattern
(+): Clindamycin RESISTANT
Uses a strip with single antibiotic of different conc. along its length
E-test/MIC on a stick
E-test/MIC on a stick is quali or quanti?
Quantitative AST → Dilution method
E-test/MIC on a stick is recommended for what type of bacteria
fastidious & aerobes
what is measured in E-test?
tapering = MIC
T/F
E-test/MIC on a stick is routinely used in the lab
F
Not routinely used; used in research
(+) result of E-test
Ellipse of growth inhibition
aka Serum Bactericidal Test
Schlichter test
Measures activity of antibiotics in patient’s own serum against the pathogen, to detect if patient is receiving effective tx for infection
Serum Bactericidal Test/Schlichter test
Screening test for carbapenemase-producing organisms
Modified Hodge Test
+ result of Modified Hodge Test
clover leaf like pattern of ZOI
