PATH5191 L8: Bacteriology QC and QAP 4/23/26

What is this?

-Product-oriented

-Inspecting, testing, and checking service/product

-Make sure it conforms to the specifications, functions as required

Internal Quality Control (IQC)

What is this?

-Process Oriented

-Preventing mistakes/avoiding problems when delivering services

-Make sure you are doing the right things, the right way.

-Preventing nonconformities/defects.

External Quality Assurance (EQA)

Is this IQC or EQA?

-Doing periodic checks to see that a horse is still in its stable

Internal Quality Control (IQC)

Is this IQC or EQA?

-Make sure that the stable doors and gate locks work properly and that all the stablehands are trained and aware of the need to close and bolt doors properly

-Training, document control and audits

External Quality Assurance (EQA)

What information does a Bacteriologist need to determine?

1) Test

2) Media

3) Temperature

4) Duration

5) Atmosphere

6) Who? When? How is the assay done?

How long does most staff training take for G2/4 Trainers and above?

>2 years, between 1 and 5 sessions

What equipment/materials were used for the Tapeworm (Taenia) segment identification procedure?

-Petri dish, glass slides, gloves

-India ink

-1ml BD insulin syringe

-Orange sticks

-Basic stereo and Olympus BX-40 light microscope

-Waste disposal bin for infectious material

What information is required on an Enteric Request Form?

1) Patient Name, Barcode, Hospital

2) Doctor's Signature and Date

3) Date and Time Collection

What is this?

1) Salmonella, Campylobacter, Shigella, and Clostridium difficile

2) Wet Prep: Parasites, Leucocytes, Erythrocytes, Charcot-Leyden crystals

3) Concentrate (Parasites)

MC&S and OCP

What bacteria species would you examine for Microscopy, Culture and Sensitivity, Ova, Cysts and Parasites (MC&S and OCP)?

-Campylobacter

-Clostridium difficile

-Salmonella

-Shigella

What wet prep would you examine for Microscopy, Culture and Sensitivity, Ova, Cysts and Parasites (MC&S and OCP)?

-Parasites

-Leucocytes

-Erythrocytes

-Charcot-Leyden crystals

What culture medium is this?

-Used for Shigella and Salmonella

-Direct and Indirect culture

-Incubate aerobically at 35°C 18-24hrs

Xylose Lysine Desoxycholate (XLD)

How would you incubate XLD medium (Salmonella or Shigella)?

-Aerobically at 35°C 18-24hrs

What culture medium is this?

-Direct culture

-Incubate at 42°C for 48 hours

-Atmosphere: Microaerophilic, 90% nitrogen, 10% CO2

Campylobacter Medium

How would you incubate microaerophilic Campylobacter medium?

-42°C for 48 hours

-90% nitrogen, 10% CO2

What information is found on a PathWest QC Report for Bacteriology specimens?

1) Expiry date/shelf life

2) Final pH and consistency

3) Physical appearance, volume and sterility

4) Performance data results

5) Incubation condition

6) Growth

7) Whether organism conforms to criteria

What is this?

-Supplies reference cultures for QC comparison in Bacteriology

-Control Organisms used for maintenance and supply of cultures

Western Australia Culture Collection (WACC)

What does WACC do in Bacteriology QC?

-Confirms identity, characteristics and chain of custody

-Culture is pure and shows the strain characteristics

-Traceable to the original seed lot

How can you confirm the identity of an organism from a WACC culture?

Use polyphasic (genotypic/phenotypic) testing

Name the order of lab maintenance for QC organisms

1) First generation: Master stock, reconstitute lyophilized ampoules

2) Second generation: STOCK cultures or WORKING cultures

3) Third generation: Subculture into STOCK cultures or WORKING cultures

What is a WORKING culture?

-Used for daily work

-Discard as determined by procedures

True or False: WORKING cultures should never be used to establish new STOCK cultures

True

What happens when stocks are taken to the limit of stability or become contaminated?

Return to Master Stock stage (first generation)

What species of Campylobacter are tested, and how long do you wait for growth?

-Campylobacter jejuni

-Campylobacter coli

-Wait for 48h

What is this antibiotic susceptibility test?

-Carrier disc is applied to a standardized lawn inoculum of bacteria

-Inhibitory zone diameter correlates to -> antimicrobial content

Disc diffusion

True or False: Control organisms need to have known sensitivity characteristics for antibiotic susceptibility testing

True

What is this automated Enteric Testing system?

-Combines Nucleic acid extraction + Thermocycling on a single platform

-Can be run in an ordinary microbiology lab

-Uses 2 controls

BD MAX PCR

True or False: You Do NOT need a clean room for BD MAX PCR, as it can be done in an ordinary microbiology lab.

True

How many controls are used for BD MAX PCR?

2 external controls used

What is this?

-CLSI

-Responsible for updating/modifying Kirby and Bauer

-Ensures uniformity of technique, reproducibility of results

-Clinical Laboratory Standards Institute (CLSI)

You are using BD MAX PCR to amplify the C. difficile toxin B gene.

Which control is this?

-ATCC® 43255, a C. difficile strain bearing the tcdB gene

-Intended to monitor for substantial reagent failure.

External Positive Control

You are using BD MAX PCR to amplify the C. difficile toxin B gene.

Which control is this?

-ATCC® 700057, a non-toxigenic C. difficile strain.

-Used to detect reagent or environmental contamination (carry-over) by tcdB amplicons.

External Negative Control

What does each BD MAX Cdiff Extraction Tube contain?

Sample Processing Control (SPC)

What is this?

-SPC

-Plasmid contains: Target DNA sequence (synthetic)

-Monitors efficiency of DNA capture, washing and elution

-Monitors DNA amplification and detection during PCR analysis

What happens if the SPC result fails to meet acceptance criteria?

-Result of the specimen: Unresolved

-Indicative of specimen-associated inhibition or reagent failure

What are some examples of viral Enteric testing?

-Adenovirus 40/41

-Astrovirus

-Norovirus

-Rotavirus

-Sapovirus.

What is this?

-S.T.A.R. Buffer

-Used for split samples

-Can bind inhibitory substances from stool

-Inactivates nucleases

-Stabilizes nucleic acids

True or False: S.T.A.R. Buffer is suitable to use with both manual and automated extraction methods

True

How often are negative controls used alongside patient specimens for enteric testing in Bacteriology?

Between every 5th patient sample

True or False: Contamination with bacteria is a greater risk than viruses

False

True or False: Contamination with viruses is a greater risk than bacterial

True

Describe the cleaning regime for Bacteriology QC

1) Contaminated hoods and benches are disinfected

2) UV: Safety cabinets, environmental testing once per month

What would you use to disinfect contaminated hoods/benches?

1) Detergent

2) 2% Sodium Hypochlorite solution

3) 70% Ethyl Alcohol

How often should you conduct UV safety cabinet environmental testing?

-Once per month

-Retest weak positives if next to a strong positive

What is this?

-A mechanism of genetic variation in viruses that involves small, continuous changes in the virus's antigens (proteins on the virus surface)

-Occur due to mutations in the viral genome.

Antigenic Drift

What virus is notorious for genetic drift?

Norovirus

How can we keep up with norovirus antigenic drift?

Use another testing method on random negative samples over a period of a month.

What is this?

-Range within which the true value of a measurement lies, considering all possible errors

Measurement of Uncertainty

Is this information qualitative or quantitative?

1) Enteric assay used to identify organism

2) RT-PCR used to produce cycle threshold (CT) value

1) Qualitative

2) Quantitative

What would you calculate for each assay?

Average, STDev, CV and measurement uncertainty

What a ±2SD value and how do we calculate them?

-Represents fixed range of acceptability

-2 standard deviations on either side of the mean

-Requires 200 minimum data points to calculate

What does it mean the routine control CT values fall within the limits of ±2SD value?

Test batch has performed at a 95% confidence level

How often are ±2SD values reviewed in a Bacteriology lab?

-Every month

-When a gross shift in control CT average is seen, an investigation is required

What is this Enteric Test?

-Fecal samples are concentrated for parasite examination.

-2 months of training before staff member is signed off

-Reference books and own collection of parasites used in aiding ID of organisms.

Mini Paraceps

What is this Enteric Test?

-Used to identify Cryptosporidia

-Positive Control organisms used in all staining procedures.

-Shows correct technique and organism appearance

Safranin/Methylene Blue Stain

What test can you use to identify the difference between invasive Salmonella Paratyphi B and non-invasive Salmonella Paratyphi B java?

D Tartrate and Slime Wall testing

True or False: Salmonella Paratyphi B and non-invasive Salmonella Paratyphi B java are both from serovar 4,5:b:1,2

True

Is this Salmonella Paratyphi B or non-invasive Salmonella Paratyphi B java?

-D tartrate is positive (+)

-Slime wall is negative (-)

Salmonella Paratyphi B (var java)

Is this Salmonella Paratyphi B or non-invasive Salmonella Paratyphi B java?

-D tartrate is negative (-)

-Slime wall is positive (+)

Salmonella Paratyphi B (invasive)

What Bacteriology tests does RCPA QAP monitor?

-Urine

-Skin/ear/eye/throat/nose

-Feces

-Respiratory

-Genital/anaerobe

-Mycobacteriology

-Difficult/blood culture/sterile sites isolates

What Bacteriology programs does RCPA QAP monitor?

-Mycology, Parasitology, Urine Dipstick Chemistry, Stat Microscopy, Molecular Diagnostics, and C.diff Detection

-VRE Screening, Pneumococci Serotyping, Haemophilus influenzae and Neisseria meningitidis, Microbiological Surveillance Culture for Endoscopes and Bronchoscopes

What RCPA QAP samples are not lyophilized?

1) AFB stains

2) Gram stains

3) Parasitology samples

True or False: Faecal, respiratory, genital, throat and skin modules for RCPA QAP are prepared without including normal flora

False

What is this?

-CAR

-Corrective Action Report

-Official recorded document

-Possible causes why we failed to detect the organism

-Changes that we would put in place to prevent errors

Who is responsible for reviewing participation in RCPA?

1) Head of Department

2) Quality Manager

3) Clinical Microbiologist

What is this?

-Responsible for providing accreditation services

-Assesses laboratories against national and international standards

-Conducts independent assessments of laboratories' processes

NATA (NATIONAL AUSTRALIA TESTING AUTHORITY)

What standards does NATA use for 1) medical laboratories and 2) NATA assessments?

1) ISO 15189

2) NPAAC

What is this?

-Develops/maintains the accreditation standards

-Advises Aus Government on pathology accreditation

-Sets standards that laboratories must meet to be accredited providers of Medicare rebateable services

NPAAC (NATIONAL PATHOLOGY ACCREDITAITON ADVISORY COUNCIL)

What is this?

-Regulates the supply, import, export, manufacturing, and advertising of therapeutic goods

-Evaluates therapeutic goods before they can be marketed

-Ensure ongoing compliance with safety and quality standards

-Recalls/issues safety alerts for risky therapeutic goods

TGA (Therapic Goods Association)