OSPE & AETCOM

OSPE 1 : Segregate the following biomedical waste in the respective colour coded bins

• Human anatomical waste

• Animal waste

• Microbiological waste

• Soiled bedsheets linens , OT gowns

• Contaminated plastic waste

• Rubber tubings

• Catheters

• Drip sets

• Glassware : test tubes , flasks , jars

• Metallic implants
Waste sharps

Broken glassware

Needles

OSPE 2 : Sterilise the following materials

AUTOCLAVE :

▣ Moist heat sterilisation

▣ Above 100 0 C

▣ Mechanism of action : protein denaturation & coagulation

▣ Used for :

▣ Culture media

▣ Plastic plates

▣ OT gowns , gloves , caps

HOT AIR OVEN :

▣ Dry heat sterilisation

▣ Above 100 0 C

▣ MOA : Damage by oxidising molecules

▣ Used for : glassware

▣ Scissors , scalpels , cotton swabs , glass syringes , OT instruments
PASTEURISATION : INSPISSATION :

• Moist heat sterilisation • Moist heat

• Milk • Below 100 0 C

▣ Below 100 0 C • Egg and serum containing media

▣ MOA : protein denaturation and • MOA : protein denaturation and

 coagulation coagulation

▣ Holder method

▣ Flash method

TYNDALLISATION : FILTRATION :

• Steam at 100 0 c • Cold sterilisation

• Koch Arnold steamer • Heat labile liquids like sera , sugar

solutions , antibiotic solutions
• Media containing sugars & gelatin
• Renders the solution only bacteria
• MOA : protein denaturation and
free , not virus free
coagulation

EMICAL AGENT MOA MATERIALS STERILISED

ylene oxide Disrupts DNA of the microorganism Cardiac catheters , implants , surgical i
heat sensitive items lijke disposable syr
petri dishes , heart lung machines

taraldehyde Disrupts bacterial cell membrane Endoscopes OT surface cleaning

h level disinfection

ohols Denaturation and coagulation of Bed pans , bp cuff , bed rails , bedside t
w level disinfectant proteins and disrupting the cell wall rub , thermometers

ium hypochlorite By removal of free sulphydryl groups Blood spill , in discarding jars , disinfec
essential for functioning of enzymes and other specimens before discarding
 ma sterilisation Damages DNA , damage by oxidising Ventilator and tonometer
molecules

OSPE 3 : Focus the given slide under low power , high power and oil immersion objective

Low power High power Oil immersion

1. Light low Medium High

Mirror Concave Concave Concave

ondensor Low Low High

perture Partially opened Partially opened Fully opened

bjective lens 10x , convex , yellow 40x , convex , blue 100x , convex , white

ye piece lens 10x 10x 10x

 tage adjustments Objective lens should Objective lens should not Objective lens should touch the sl
not touch the slide touch the slide

il interface No No Yes

Magnification 100x 400x 1000x

OSPE 4 : Collect a nasopharyngeal swab from a patient suspected of infection

1. Perform hand hygiene and wear personal protective equipment

2. Take informed consent of the procedure to be performed

3. Stand on right side of the patient and tilt their head backwards

4. Grasp the swab like a pencil towards the shaft

5. Place the swab at the bottom of the nostril. Travel along the floor of the nose , which
corresponds to the hard palate .

6. On reaching the posterior nasopharynx , stop and rotate the swab several times and remove
the swab slowly.

7. Open the collection tube containing transport media and insert the swab into the tube .

8. Label the container as “BIO HAZARD “.

9. Transport the specimen to the laboratory as soon as possible .

 OSPE 5 : Collect a throat swab from a patient suspected of infection

1. Perform hand hygiene and wear personal protective equipment .

2. Take informed consent of the procedure to be performed .

3. Stand on right side of the patient and tilt their head backwards.

4. Ask the patient to open their mouth and protrude their tongue forward .

5. Use a tongue depressor to hold the tongue in place.

6. Without touching sides of the mouth use a sterile flocked swab to swab the posterior
nasopharynx and the tonsillar arches.

7. Open the collection tube containing transport media and insert the swab into the tube and
label it as BIOHAZARD .

8. Transport the specimen to the laboratory as soon as possible .

OSPE 6 : Instruct a patient suspected of pulmonary tuberculosis to collect sputum sample

1. Rinse or gargle mouth with betadiene solution .

2. Use a wide mouthed container , open the container .

3. Hold the container to your mouth with the lips inside .

4. Take a deep breath and cough out the sputum into the container .

5. Close the container tightly and send to the laboratory with a detailed requisition form .

Points to be remembered :

• The sample should have mucoid consistency.

• Early morning sample is the ideal one .

• Any delay in sample transport , it should be stored in the refrigerator for a maximum
period of 24 hours .
 • Sample should be minimum 1 ml .

OSPE 7 : Instruct a healthcare worker to collect intravenous blood sample of a patient for
culture and sensitivity

1. Take informed consent of the patient.

2. Wear double gloves .

3. Tie a torniquette above the cubital fossa.

4. Ask the patient to make a fist .

5. Clean the cubital fossa first with povidone iodine and then with 70% ethyl alcohol . Allow
it to air dry.

6. Locate the median cubital vein and localise it with the thumb and index finger .

7. With the beveled edge of the needle upwards at an angle of 45 0 prick the vein.

8. Release the torniquette and the fist .

 9. Withdraw 3-5 ml of blood in children and 8-10 ml of blood in adults and transfer the
blood directly into the blood culture bottle containing the Brain – Heart Infusion Broth .
Label the bottle as “HIGH RISK “.

10. Place a cotton swab dipped in alcohol over the prick area while removing the needle .

11. Arm should be folded .

12. Discard the needle in the white bag and the syringe and needle cap into the red bag.

13. For tests like CBC 2 ml of blood is to be collected in a EDTA tube .

14. For tests like RBS , RFT , SR.ELECTROLYTES , CRP , ESR , HIV , HBsAg , VDRL ,
Dengue NS1 Ag , etc 2 ml of blood should be collected in a plain tube .

OSPE 8 : Instruct a ______ patient to collect urine sample for culture and sensitivity

FEMALE PATIENT :

1. Rinse the area with soap and water

2. Clean it with a sterile gauze piece

3. With both labia apart allow the first few drops of the urine to drain

4. Take midstream clean catch urine preferrably early in the morning

5. Collect the urine into wide mouthed , sterile , leak proof container

6. Transport it directly to the laboratory after proper labeling

7. If delay in transport , preserve it at 4-6 0 c

MALE PATIENT :

▣ Wash hands and retract prepuce before collecting the sample

▣ Clean it with sterile gauze

▣ Further steps are same as point no 4-9 in female urine collection

INFANTS :

❖ Bag urine method

 ❖ Pad urine pack

❖ Clean catch / midstream urine in a sterile container

❖ Suprapubic aspiration

CLEAN CATCH URINE :

• Wash hands thoroughly and wear gloves

• Clean the skin around the genital area with soap and water or cleaning wipes

• Keep watching and make sure the urine container remains clean

• Catch the urine away from your child’s skin

CATHETERISED PATIENTS :

▣ If the catheter is freshly placed the sample can be collected directly from the catheter

▣ Make a note to disinfect the urethra before inserting the catheter

▣ DO NOT collect the urine from the urine bag

▣ Disconnect the urine bag from the catheter , allow few drops of urine to drain and then
collect the urine in a wide mouthed sterile , leak proof container

OSPE 9 : Instruct a patient of suspected cholera to collect stool sample for culture
and sensitivity

▣ Container : sterile , leak proof , puncture proof , wide mouthed , of 40ml capacity
▣ With appropriate labeling transport the sample to the laboratory within 2 hours of
collection

▣ In case of delay in transportation send the sample in transport media like phosphate buffer
saline , cary blair medium , VR medium , stuart’s medium , alkaline peptone water

▣ Sample can be preserved in refrigerator or ice packs in case of any delay in transportation

▣ Sample should never be collected in bed pan

OSPE 10 : Collect the hair , nail clippings and skin scrappings for confirmation of
dermatophytosis in a clinically diagnosed case

▣ Container : clean screw top tube or black paper

▣ For nail & skin wipe the are with 70% alcohol

▣ Hair – collect with intact shaft by plucking using forceps with follicle

▣ Nail – clipping from the nail bed

▣ Skin – scrape skin at leading edge of lesion with a scalpel blade

▣ Transport within 24 hrs at room temperature

OSPE 11 : Collect a sample from a wound/ pus for culture and sensitivity

▣ Clean the contaminating material such as slough , debris , dried exudate and dressing
residue with tap water , sterile saline or by debridement

▣ Use dacron or rayon swabs preferably over cotton swabs

▣ The swab should be moved across the wound surface in a zig-zag pattern , at the same
time as being rotated between the fingers

▣ Place the swab back into transport container , label it and send to the laboratory

▣ NOTE : If the discharge is dry kindly moisten it with normal saline

 OSPE 12 : Demonstrate the steps of HAND RUB & HAND WASH

HAND WASH HAND RUB

1. Wet hands with water and apply ▣ Apply about 3 ml of the alcohol rub
enough soap to cover all hand onto your cupped hands
surfaces
1. Rub hands palm to palm
2. Rub hands palm to palm
2. Right palm over left dorsum with
3. Right palm over left dorsum with interlaced fingers and vice versa
interlaced fingers and vice versa
3. Palm to palm with fingers interlaced
4. Palm to palm with fingers interlaced
4. Back of fingers to opposing palms
5. Back of fingers to opposing palms with fingers interlocked
with fingers interlocked
5. Rotational rubbing of left thumb
6. Rotational rubbing of left thumb clasped in right palm and vice versa
clasped in right palm and vice versa
6. Rotational rubbing backwards and
7. Rotational rubbing backwards and forwards with clasped fingers of
forwards with clasped fingers of right hand in left palm and vice
right hand in left palm and vice versa
versa
7. Rub each wrist with opposite hand
8. Rinse hands with water
8. Wait until the product has
evaporated and the hands are dry
 AETCOM 1 : Instruct a wardboy regarding biospill management

❖ Raise an alarm and contain the area

❖ Wear PPE

❖ Use plastic scope to remove any broken pieces of glasses

❖ Contain the spill with absorbent granules

❖ Remove the solidified waste material using the scope and scrapper and carefully dispose
off all contaminated material into the infectious waste(yellow) bag

❖ Add chlorine disinfectant 🡪 5% sodium hypochlorite

❖ Contact period: 30 min –45 mins

❖ for large spill >10cm = 1:10 dilution

❖ for small spill <10cm = 1: 100 dilution

❖ Use disposable wiping clothes

❖ Place all the items in the yellow bag including PPE

❖ Close the biohazard bag

❖ Hand hygiene

❖ Inform admin about the biohazard spill

❖ Record the incident in the incident register book

AETCOM 2 : Advice a HCW with needle stick injury in a complete and correct
sequence in a simulated setting

1. First aid

1. Allow free bleeding

2. Do not squeeze
 3. Do not put finger in the mouth

4. Wash the area with running tap water

5. Do not use spirit for cleaning

2. Inform the Infection control committee, concerned medical staff posted in the ART/ ICTC
centre.

3. Take first dose of PEP for HIV within 2- 72 hrs

4. Check the status of the patient and the HCW for HIV , HBsAg & HCV

5. Complete and submit needle stick injury form

6. Follow the needle stick injury policy – for HIV

- PEP 28 Days

- primary TL + LR regimen

NSI

if rapid HIV if rapid HIV

negative positive
no PEP PEP
no testing for HBV & HCV testing for HBV & HCV
advice to take vaccine vaccination
 If HBsAg / HCV positive

Completely
Completely vacccinated
vaccinated Not vaccinated
Titre < 10 micu/ml
Titre > 10micu/ml Source +
Source positive
Source +
HBsAg 1st dose
No PEP within 7 days
required followed by 3
doses

7. Follow up testing

• -- HIV 🡪 6 WEEKS , 3 MONTHS & 6 MONTHS

• -- HBV & HCV 🡪 6 MONTHS

AETCOM 3 : Demonstrate respect to patient sample

Case scenario 1 (Rejection due to improper transport):

• Sequestrum from chronic osteomyelitis case was debrided and sent for
culture and sensitivity.
• The sample was rejected by the laboratory mentioning that it was received
in formalin, hence unsuitable for culture.
• There is no more sample available for culture now

▣ Competency : this is a case of rejection due to improper transport

▣ Standard procedure of collection and transport : two types of samples can be collected
from a case of osteomyelitis 🡪 pus and sequestrum
▣ In the above case sequestrum has been collected which should ideally be transported in
normal saline ( 0.85%) or thioglycollate broth as the sequestrum yields anaerobic
organisms more commonly .

▣ Formalin kills the bacteria , hence samples received in formalin are not suitable for
microbiology culture . Hence this sample needs to be rejected.

▣ Ethical issues : Patient has to undergo debridment again which is time consuming and then
3 more days he needs to be hospitalised as the culture takes minimum 3 days , ultimately
longer hospital stay , also inaccurate reports as treatment might already have been started.
Also patient may not give consent for repeated procedures.

▣ Medicolegal issues : this may account for negligence leading the clinician and related
people behind the bars , as the clinician is unaware and uninformed about the standard
operating procedures.

Case Scenarios 2 (Specimen did not reach laboratory):

• A critically ill 5-year-old child’s CSF report is awaited for 3 days. On enquiry
laboratory says it did not receive the sample. On further probing it was found that
the nursing staff had kept the small bottle with the sample in his pocket and
mistakenly taken it outside the hospital and had dropped it somewhere, and did not
submit it to the laboratory for testing. Now, the baby needs to undergo lumbar
puncture again, results may not be the same as antibiotics are given and need to wait
for some more days for the culture report.

▣ Specimen : CSF 🡪 Priority 🡪should be processed within 2 hours of collection

▣ Container : sterile , leak proof

▣ Sample should be collected before starting antibiotics

▣ Sample should not be exposed to direct sunlight nor it should be freezed

▣ In case of any delay the specimen should be kept at room temperature

▣ Medicolegal issue : negligence of the staff

▣ Ethical : painful procedure for the baby , longer hospital stay , inappropriate reports if
sample is sent after starting antibiotics

Case Scenarios 3 (Misguided report due to inadequate information in requisition

form):

• Urologist calls laboratory to discuss about “Insignificant bacteriuria” culture report

of a pyelonephritis patient. He says it was a percutaneous nephrostomy sample and
asks for the organism and antimicrobial sensitivity. Microbiologist says it was written
as urine sample on the request form, some gram-negative bacillus had grown and the
count was less than 10,000 CFU/mL, so it was thought to be a periurethral
commensal and the isolate was discarded, and hence further testing cannot be done

▣ Sample sent :urine from percutaneous nephrostomy

▣ Requisition says it is a urine sample

▣ Percutaneous nephrostomy is an invasive procedure to collect urine directly from the

kidneys

▣ Full plate of blood agar without intermittent heating should be streaked in such a sample

▣ Any number of colony count is significant in PCN

▣ THEREFORE PROPER LABELING OF THE REQUISITION IS IMPORTANT

▣ Medicolegal issue : inappropriate reports , delayed reports and inefficient management of

the patient

▣ Ethical issue : painful procedure , repeat billing needs to be done for another sample ,
longer hospital stay , patient may deteriorate

Case scenario 4 : specimen kept at wrong place :

junior resident gets angry and yells at the patient on noticing a stool sample kept on
bedside .............

▣ In our setup we have a separate sample collection room (30) nearby to the laboratory (31)

▣ Three different sections are made one each for samples going to biochemistry , pathology
and microbiology

▣ From the microbiology set the samples are segregated

▣ Vacutainers go to the serology section

▣ Blood in BHI broth , sputum, pus , fluids from sterile sites , stool for culture and
sensitivity goes to bacteriology section

▣ Stool for parasite examination should go to parasitology section

▣ Ocular samples for koh , skin scrapping , nail , hair for fungal culture should go to
mycology section

▣ Sputum for AFB should be sent to CBNAAT centre (105) beside nandini milk parlour

▣ Medical issue : as in this case the stool sample is kept in blood sample tray , there are
chances of contamination of both the samples and also there is risk of rejection or
discarding of the sample

▣ Ethical issue : lack of communication between doctor and patient and the attitude of the
doctor towards the patient

Case Scenarios 5 (Rejection due to improper collection):

• A suspected pulmonary tuberculosis patient, who would travel 30 km from his

village to the private hospital with the attached laboratory in the city, had submitted
spot sputum sample the previous day and an early morning sample today for acid-
fast staining. Reports of both the samples mentioned “many epithelial cells suggestive
of excessive salivary contamination. Repeat with the proper sample”. Blood culture
was also collected from the patient by the clinical team, the result of which came as
contaminated blood culture specimen with patient’s skin flora

▣ Many epithelial cells on gram’s stain suggests that the sample is saliva

▣ Contamination in blood culture suggests that the skin wasnt adequately disinfected while
collecting the sample

▣ Refer to sputum and blood collection protocol discussed previously

▣ Medicolegal issues : delayed and inappropriate or wrong reports

▣ Ethical issues : financial – travelling expenses and then the tests expenses
 Case Scenarios 6 (Sample collected for culture and sensitivity in unsterile container):

• Paired blood specimen (5 mL each) was sent to the laboratory in two vacutainers
for blood culture. The laboratory rejected the specimen. The patient screams that he
cannot allow to draw another set of blood specimen for investigation

▣ Sample for blood culture should be collected in blood culture bottles containing BHI only

▣ Vacutainers contain anticoagulants which interfere with the growth of the bacteria

▣ Also the sample sent is inadequate for culture

▣ Ethical issue : painful punctures , if blood disorders present may aggrevate the patients
condition , more risk of bloodborne infections

Case scenario 7 : rejection due to lack of patient information

▣ If name has not been mentioned there may be exchange of results

▣ Age : in children below 2 yrs of age any isolate in the stool sample is significant , whereas
the isolate becomes a commensal > 2 yrs of age

▣ Sex : urine sample collected from a female patient has a risk of vaginal contamination with
candida . Staphylococcus saprophyticus isolated from Urine of female patient is a
pathogen whereas in a male patient it is a contaminant

▣ Travel history : > 2yrs with history of travel to endemic areas any isolate in the stool
sample is significant

▣ Date and time of sample collection : motility of the organisms cannot be visualised if the
specimen is transported after 2 hrs of collection

▣ Patient id and the MIC number is important for result to be generated

▣ Processing of different specimens differs . Therefore it is important to mention the type of

specimen collected and the test requested

▣ As the laboratory is busy all the time with less manpower , the requisition forms are
generated to ease the work of both the clinician and the microbiologist

▣ Ethical issue : due to rejection of sample , the patient has to get fresh billing again and the
clinician should collect fresh specimen again
 Case scenario 8 : rejection due to mismatch of name

▣ The laboratory cannot accept such specimens considering the sample might have been
misplaced or exchanged with some other form

▣ Ethical issue : once rejected , rebilling needs to be done with appropriate requisition form

Case Scenarios 9 (Prioritizing a sample requiring immediate processing and

reporting over the others)

• In the midnight, the Microbiology laboratory receives three specimens (urine,

sputum, CSF) from a patient for culture. The technician was already processing a
huge load of investigations, therefore he informed the clinical team that these
specimens can only be processed on the next day

▣ In this case the CSF should be processed immediately and the sputum and urine samples
needs to be refrigerated as there is delay in processing

▣ Fragile organisms like Neisseria and Influenza in the CSF dies when refrigerated