Specimen collection, processing, and handling are important for accurate microbiological testing. Key points include collecting specimens before initiating antimicrobial therapy, using adequate volumes, and expediting transport. Proper collection methods and transport media are needed to preserve specimen integrity. Timing of collection depends on the suspected pathogen. Adequate labeling and packaging is required for referral testing. Universal precautions must be followed when handling all specimens.

1. Specimen Collection,
Processing and Handling

2. KEY POINTS
• Collect specimens from site of infection prior
to initiation of therapy.
• Collect adequate volume of sample for testing
required.
• For bacterial, fungal and viral cultures submit
tissue, fluid/aspirate if possible as these are
always superior to a swab specimen.

3. KEY POINTS
• Use required collection and transport
materials to preserve specimen integrity.
• Communicate clear orders and source
information.
• Expedite the transport of specimens to the
laboratory and do not allow them to sit in
collection areas.

4. GENERAL PRINCIPLES
• TIMING OF SPECIMEN COLLECTION
– Should be collected at a time when the likelihood
of recovering the suspected pathogen is greatest.
• RECOVERING VIRUSES : greatest in the acute phase of
the illness.
• RECOVERING BACTERIA : ideally be collected before
antimicrobial therapy is started.

5. TIMING OF SPECIMEN COLLECTION
• ENTERIC INFECTION
– Organisms are in greater number during
acute/diarrheal stage.
• TYPHOID FEVER
– Blood culture in the 1st week of infection.
– Stool/Urine culture on the 2nd week
– Serological tests in the 3rd week

6. SPECIMEN VOLUME
• MUST BE ADEQUATE for performance of the
microbiological studies requested.
• IF INSUFFICIENT : notify nurse/physician for
additional sample/to prioritize the requests.
• IF A SWAB :
– use polyester-tipped swab on a plate shaft
– avoid calcium alginate swab for viral culture
(inactivates herpes simplex virus)
• cotton toxic :Neisseria gonorrhoeae
• Wood toxic : Chlamydia trachomatis

7. SPECIMEN VOLUME
• SWAB : not optimal for detection of anaerobes,
mycobacteria/fungi, do not use.
• An actual tissue sample of fluid aspirate is
always superior to a swab specimen for the
recovery of pathogenic organisms.

8. SPECIMEN COLLECTION
• Obtained from the site of infection with
minimal contamination from adjacent tissues
and organ secretions and collected in a sterile
container.
• LABEL : name & identification number of the
patient, the source of specimen, date and time
of collection.

9. SPECIMEN TRANSPORT
• Placed in a biohazard bag and transported to the
laboratory as soon as possible.
• IF DELAY IS UNAVOIDABLE : refrigerate to prevent
overgrowth of microbiota.
– Urine, sputum, other respiratory specimens, stool.
– Specimens for detection of Chlamydia
trachomatis/viruses.
• Cerebrospinal fluid (CSF) and other body fluids,
blood, specimens collected for recovery of
– Neisseria gonorrhoeae : room tempt., if refrigerated
adversely affects recovery of potential pathogens
froms these sources.

10. UNACCEPTABLE SPECIMENS
• SPECIMENS
– Received in formalin
– In containers from which
the sample has leaked
– That have been
inoculated on to agar
plates that have dried
out/oudated
– Contaminated with
barium, dye/oily
chemicals
• 24hr sputum collection
• Foley catheter tips.
• Duplicate specimens
(except blood cultures)
received in a 24hr
period
• Blood catheter tips
submitted for patients
w/o concomitant
positive blood culture.

11. SPECIMEN REJECTED FOR ANAEROBIC
CULTURE:
• Gastric washings
• Urine other than suprapubic aspirate
• Stool (except for recovery of Clostridium difficile )
– for epidemiologic studies/ for diagnosis of bacteria
associated with food poisoning.
• Oropharyngeal specimens (except deep tissue samples
obtained during a surgical procedure)
• Sputum
• Swabs of ileostomy/ colostomy sites.
• Superficial skin specimens.

12. UNIVERSAL PRECAUTIONS
• Must be followed when handling all specimens
• APPROPRIATE BARRIERS : to prevent exposure of
skin & mucous membranes to the specimen
• PPE
– Gloves and a lab coat must be worn at all times
– Masks, goggles and impermeable gowns/ aprons must
be worn in situation which risk of splashes/ droplet
formation
• Open specimen container in serological safety
cabinet.

13. REFERRAL TESTING
• MUST BE PACKAGED
– Shipping specimens/cultures to reference lab
– Dangerous good shipping guidelines (IATA website)
• SPECIMENS : not more than 40 ml
• Cultures of bacteria and fungi
– Grown in solid media and tubes
• Cap sealed with waterproof tape

14. REFERRAL TESTING
• Sufficient packing (1°, 2°)
– Leak proof
– Prevent from breaking
– Individually wrapped/separated
• Itemized list of contents enclosed,
marked/labeled properly
• Labeled with an official label
– Address, contents, name, telephone number of
person responsible for shipping.

15. CLINICAL SPECIMENS
• BLOOD & BLOOD COMPONENTS
• THROAT & NASOPHARYNGEAL DISCHARGES
• SPUTUM
URINE
• CEREBROSPINAL FLUID (CSF)
• STOOL
• GENITAL DISCHARGE
• WOUND DISCHARGE

16. BLOOD
• Detect blood-borne pathogens
• Identify bacteria responsible for
– Bacteremia
– Sepsis infections of the native and prosthetic
valves
– Suppurative thrombophlebitis
– Mycotic aneurysms
– Infections of vascular grafts
• Collect before beginning antimicrobial therapy

17. BLOOD
• When any one/ a combination of the following:
– Fever (38°C or greater)
– Hypothermia ( 36°C or lower)
– Leukocytosis (especially with a left shift)
– Granulocytopenia
– Hypotension

18. BLOOD : SPECIMEN COLLECTION
• Phlebotomy technique
– Minimize contamination with skin flora
– 1st: Skin cleaned with 70% alcohol
– 2nd: Apply 1-2% iodine solution, an
iodophor/chlorhexidine; dry for 1-2minutes prior
to venipuncture.

19. BLOOD : APPROPRIATE TIMING FOR DETECTION
OF BACTEREMIA & FUNGEMIA
• Optimal time to collect : just before a chill (not
predictable)
• Collected after the onset of fever and chills
• Needle and syringe
– Without changing needles
– Injected directly into bottles of culture media
– Inverted several times to mix
– Transported to lab at room temp. as soon after
collection is possible.
– SHOULD NOT BE REFRIGERATED

20. BLOOD : SPECIMEN VOLUME
• Bacteremia in adults (CFU/ml) = low
– 20-30 ml of blood per culture set = strongly
recommended.
• Infants and children = higher concentration of
microorganisms
– 1-5 ml of blood per culture = adequate

21. BLOOD : SPECIMEN DRAWS
• Number of blood specimens = based in the nature
of bacteremia
– Transient, Intermittent or Continuous
– TRANSIENT BACTEREMIA
• Follows manipulation of a focus of infection, instrumentation of
a contaminated mucosal surface or a surgical procedure in a
contaminated site; also in the course of many systemic and
localized infections.
– INTERMITTENT BACTEREMIA
• Associated with an undrained abscess
– CONTINUOUS BACTEREMIA
• Hallmark of intravascular infection

22. BLOOD : SPECIMEN DRAWS
• 2-3 20 ml blood samples drawn over 24hr
period, aerobic and anaerobic blood culture
bottles (sufficient)
• 4 blood cultures within 24hr = yielded more
• Suggested 30-60 min time interval for the first 2
sets, another 1-2 sets drawn (remaining 24hr)
• If antimicrobial therapy is deemed urgent, collect
before therapy, 2 separate sites within few
minutes.

23. BLOOD : SPECIMEN DRAWS
• Frequent blood culture contaminants but also
true pathogens:
– Coagulase – negative Staphylococci
– Viridans Streptococci
– Corynebacteria
– Bacillus spp. , Propionbacterium spp.
• 2 sets of blood cultures per febrile episode
recommended for differentiation (present in only
1 bottle = contamination)

24. RECOVERY OF MICOORGANISMS
• Factors that may impede recovery of pathogens:
– Antibodies
– Complement
– Phagocytic WBC
– Antimicrobial agents
• Approaches :
– Dilute blood specimen in broth medium in a 1 : 10
ratio = optimal neutralization of the serum
bactericidal activity.

25. CONT..
• Incorporating 0.02-0.05% sodium polyethanol
sulfonate in the blood culture medium = inhibits
– Coagulation
– Phagocytosis
– Complement activation
– Inactivates aminoglycosides
• Other methods:
– Adding penicillinases to broth media to inactivate
penicillins
– Using antibiotic – absorbent resins and lysis –
centrifugation system

26. MANUAL BLOOD CULTURE
• CULTURE BOTTLES
– Examined daily for 7 days for evidence of growth
– Indicated by turbidity, hemolysis, gas production,
discrete colonies/combination of these.
• If growth +
– smear, stain with Gram stain,
– read subculture + broth to appropriate agar media
• Routine subculture of macrospically negative –
broths performed after 6 – 18hrs of inoculation;
anaerobic bottle : unnecessary

27. CONT..
– ADVANTAGES : low cost, low contamination rate
– DISADVANTAGE : time consuming process of routine
bottle examination and subculture
BIPHASIC SYSTEM
– Consists of a broth medium in a bottle to which a
chamber containing agar media on a paddle is
attached.
– Colonies on the agar ate used for identification and
susceptibility testing.

28. CONT..
LYSIS – CENTRIFUGATION SYSTEM
– Consist of a tube containing reagents that inhibit
• coagulation and the complement cascade
• lyse blood cells
• provide a cushion for the microorganisms during
centrifugation.
– Blood added to tube
• Inverted several times, prevent from clotting
• Transported ASAP
• Centrifuged 30 min at 3000x g
• Discard supermatant
• Sediment vortexed and plate to agar

29. CONT..
– ADVANTAGES :
• excellent recovery of Staphylococcus aureus, some
Enterobacteriaceae, and fungi (best for molds, Histoplasma
capsulatum)
• Direct availability of colonies for identification and suspectibility
testing
• Ability to carry out quantitative cultures
• FLEXIBLE : inoculate to special media (Legionella &
mycobacteria)
– DISADVANTAGES :
• Labor – intensive
• Less likely recover Steptococcus pneumoniae , Haemophilus
influenzae/ anaerobs
• Increased risk for contamination

30. HANDLING OF SPECIMEN
• Specimen can be placed in transport medium:
– TRANSGROW : Neiserria gonorrhoeae
– AMIES : Fastidious organisms
– CARY & BLAIR/STUART’s : Fecal specimen
• Urine may be refrigerated but it must be
examined within 24hrs
• Stool and sputum could also be refrigerated
but it must be examined within 2 – 3 hrs.

31. • CSF for bacterial culture :
– Incubate for not more than 12 hrs.
– Stand at room temp not longer than 1 hr.
– DO NOT REFRIGERATE : some organisms are
sensitive to low temp.
• Neisseria meningitidis, Haemophilus influenzae
• CSF for viral culture :
– Refrigerate immediately
– If held for more than 24hrs, freeze specimen at -
70°C

32. BLOOD
• GENERAL CONSIDERATIONS :
– Collect blood during febrile episode
– During a chill
– TIMING = + BC depends on the pathogenic process
of the organism
– Obtain 2 simultaneous blood cultures in 2 sites
(right and left arm)
• Single negative blood culture doe not rule out
bacteremia.
– Collect blood aseptically
– Ideal volume is 10 ml.

33. • Use anticoagulant (e.g. SPS)
• If Px is on penicillin, administer penicillinase
– Not effective against methicillin, cloxacillin, nafcillin
• Hold for 2 weeks before reporting for negative
– 21 days for Brucella spp.
• Indications of (+) blood culture :
– Turbidity
– Hemolysis
– Colony or pellicle formation
– Presence of gas or bubble

34. • MEDIA USED :
– Trypticase Soy Broth
– Brain – Heart Infusion
– Columbia Broth
– Castaneda Broth
– Brucella Broth
– Thioglycollate Broth
• ANTICOAGULANT :
– SODIUM POLYANETHOL SULFONASE
• Neutralization bactericidal effect of serum
• Prevents phagocytosis
• Inactivates some antimicrobial agents

35. BLOOD COMPONENTS
• Organisms found in fatal transfusion reactions
are psychrophilic
• Gram negative bacilli
– Pseudomonas spp. EXCEPT Pseudomonas
aeruginosa
• PLATELET

36. THROAT & NASOPHARYNGEAL
• Alpha strep (abundant) but Group A
strep.(common pathogen)
• Culture must include anaerobic conditions for
Beta strep.
• MEDIA
– Haemophilus influenzae : CAP or BAP w/Staph
– Neisseria meningitidis : CAP /Thayer Martin
– Bordetella pertussis : Charcoal cephalaxin

37. SPUTUM
• SUITABLE Sx :
– < 10 epithelial cells & >25 pus cells
– Early morning sputum
• PROCEDURE :
– Make a direct smear for GS & AFS
– Do concentration technique (ex. NaOH)
• Free bacteria by dissolving fats and mucus
– Culture

38. URINE
• Container : sterile wide mouth glass or plastic
jar with tight – fitting lids.
• Method :
– SUPRAPUBIC ASPIRATION (IDEAL)
– CATHERTERIZATION/CYSTOSCOPY
– CLEAN CATCH MIDSTREAM
• Collection : early morning
• Processing : within 2grs after collection

39. URINE
• GS of uncentrifuged Sx for rapid screening of UTI
• Presence of numerous squamous cells indicate
vaginal/ urethral contamination
• PROCEDURE :
– Inoculate the Sx in BAP, EMB, Mac using a calibrated
loop (0.01 or 0.001 mL)
– Incubate overnight
– No growth for the 1st 24hrs, continue for 24hrs more
before reporting negative.

40. Actual # of colonies X Calibrations of loop = # of CFU/mL
100,000 bacterial CFU/mL (indicate infection)
• POUR PLATE METHOD :
– Prepare 1 : 100 dilution of urine w/ sterile H20
– Transfer 0.1mL of the solution to the Petri dish
– Add agar and mix well
– Incubate for 18 – 24hrs
– Count colonies and multiple by 100
– Report result in bacteria/mL

41. CEREBROSPINAL FLUID
• PROCEDURE
– Centrifuge Sx; make a smear for GS and India ink
– Culture Sx
• Haemophilus influenzae type B
• Neisseria meningiditis
• Steptococcus pneumoniae
• MEDIA
– TSB/thiodlycollate
– BAP for Gram (+) cocci
– CAP for Gram (-) cocci
– EMB/Mac for Gram (-) bacilli

42. STOOL CULTURE
• Ideal Specimen :
– Freshly collected stool on early stage of disease
– Rectal swab may be used
• Amount : 1-2 grams
• Container : Clean, wide mouth with lid
• Transport time : 2hrs after collection
• Transport medium : Cary Blair

43. STOOL CULTURE
• PROCEDURE
– Put rectal swab in enrichment broth or transport
medium
– GS is NOT usually done but helps in identifying
etiologic agents
• Gram + cocci in clusters (Staphylococci)
• Gram – comma-shape bacilli (Vibrio)
• Gram + bacilli in large numbers
• Gram – bacilli

44. • 1st day
– Inoculate the Sx and incubate it overnight
– MEDIA :
• Differential (EMB, Mac)
• Selective (SSA, HEA, XLD)
• Enrichment (Selenite F, APW)
• 2nd day
– Check diff’l media for LF and NLF
– Subculture and do biochemical tests
– If there’s no growth, inoculate culture from the enrichment
medium into EMB or Mac
• 3rd day
– Note the patterns of biochemical reactions
– If suggestive of Salmonella Shigella, Vibrio DO serological typing
– If growth occurs after doing step 2 of 2nd day, subculture into
biochemical test media
– Incubate overnight and perform first step of day 3.

45. EXUDATES & TRANSUDATES
• Wounds
• Boils
• Abscesses
• Ulcers
• Granules
• Rash
• Gastric Aspirates
• Tissues
• Eye discharge
• Ear discharge
• Effusions
• Endocervical
• Urethral
• Anorectal discharge

46. EXUDATES & TRANSUDATES
• Discharge/ Fluids : best to aspirate
– Dry wound : moisten swab w/ NSS before collecting
– Skin lesion : remove crust of pustule/vesicle cap then
swab lesion (Tzanck smear)
– Endocervical : use swab
– Urethra : use swab or scrape mucosa of anterior
urethra
– Anorectal : insert swab about 4-5cm. Into the anal
canal

47. COLLECTION
• Aspirates : sterile vial
• Ulcerative lesions : biopsy w/o preservations
• Irrigation, intravenous : sterile vial
• Intra-arterial Catheter tips : sterile vial
• Swabs : 2 pcs in sterile tube
• Fluids : syringe w/ sterile rubber stopper
• Corneal scraping : direct inoculation

48. GENITAL DISCHARGE
• SPECIMEN
– Cervical (female)
– Urethral (male)
– Rectal & throat swabs
• STD caused by :
– Treponema pallidum
– Neisseria gonoreheae
– Chlamydia trachomatis
– Candida albicans
– Gardenerella vaginalis
– HSV (Herpes Simplex Virus)

49. SUCCESSFUL RECOVERY OF ETIOLOGIC AGENTS
DEPENDS ON:
• Advance planning
• Collection of appropriate and adequate
specimen
• Correct packaging and rapid transport to the
laboratory
• Ability of the laboratory to accurately perform
the diagnostic tests

50. GENERAL GUIDELINES FOR SPECIMEN
COLLECTION
• Obtain specimens before treatment
• Collect material from the appropriate site
• Obtain material for culture during the acute
stage of the illness
• Collect sufficient quantity
• Transport the specimen immediately
• Label all specimen
• Specimens should be accompanied with a
request form

51. CLINICAL SPECIMEN FOR BACTERIAL CULTURE
• Blood
• CVD
• CSF
• Respiratory
• Urine
• Transudates/Exudate
s
• Feces/Rectal swab
• effussions

52. CRITICAL TIME FOR DELIVERY OF SPECIMEN OF
THE MICROBIO LAB
SPECIMEN
DELIVERY TIME
Respiratory
(sputum, throat culture) 1HR
Gastrointestinal
(stool, rectal, swab etc.) 1HR
Blood culture 1HR
Anaerobic 30 MIN
Cerebrospinal Fluid (CSF) IMMEDIATELY
Other Fluids IMMEDIATELY

53. CRITICAL TIME FOR DELIVERY OF SPECIMEN OF
THE MICROBIO LAB
SPECIMEN DELIVERY TIME
Urine 1HR
Wound, skin & soft tissue 30MIN
Fungal 1HR
Mycobacterial 1HR
Chlamydia 1HR
Except for CSF, body fluids specimens should be
delivered w/in 30mins. – 1hr to the lab.

56. VARIABLES AFFECTING BLOOD CULTURES
• Number of Collections : 2-3 collections/ 24hrs
• Volume of Blood
YOUNG CHILDREN : 1-2mL in 20mL broth (1:10 to 1:20)
ADULTS : 5-10mL in 50mL broth (1:5 to 1:10)
• Broth Medium : TSB or BHI
• Normal Bactericidal Properties of Blood
• Sodium PolyanetholsulFonate (SPS): 0.025%
Gelatin : 1%
Agar : 0.1%

57. COMPONENT OF BLOOD CULTURE BROTH
1 % Gelatin
• enhance growth of Neisseria meningitidis
0.1 % Bacto-agar
• enhance growth of anaerobic organism
• anticoagulant
0.025 % Sodium Polyanetholsulfonate (SPS)
• inhibit activity of complement & lysosyme
• prevents phagocytosis
• inactivates therapeutic concentration of
aminoglycosides

58. BLOOD CULTURE
Incubation Temperature : 35°C – 37°C
Incubation Time : 7 DAYS
Subculture on BAP & CA : after 14 – 17hrs
3days, 5days, 7days
Note: BAP & CA should be incubated at 35°C - 37°C
under CO2 enhanced atmosphere.

59. COLLECTION & TRANSPORT OF CSF
Container  Dry Sterile screw caps
Volume of sample  3 to 4 mL
culture others
Transport time  immediately w/o delay

60. CSF
• The specimen should be taken before
antibiotics are administered.
• Antigen tests may give a specific diagnosis
even after antibiotic treatment.
• CSF should always be taken from patients with
suspected purulent meningitis, regardless of
antibiotic treatment.

61. CSF SPECIMEN
• Volume : 0.5 – 5.0mL
• Time of Collection : first week of illness
– 1st tube : cell count and differential staining
– 2nd tube : Gram’s stain and culture
– 3rd tube : protein, glucose and special tests

62. Suitability of Sputum for Culture
Sputum Classification Based on WBCs & Squamous
Epithelial Cell Densities
Cell numbers per x 100 (low power) field
Group WBCs Epithelial Cells
6 <25 <5
5 >25 <10
4 >25 10-25
3 >25 >25
2 10-25 >25
1 <10 >25
“Only sputum samples in categories 4-6 should be
cultured”

63. PROCESSING OF CSF

64. TRANSPORT MEDIUM
• Stuart
• Amies
• Cary Blair