This document provides guidance on proper specimen collection, transport, and aseptic technique in microbiology. It discusses the importance of collecting the right specimen from the right patient at the right time and transporting it properly to the laboratory. It provides details on proper collection and transport of various specimen types from different sites of infection, including respiratory, urinary, genital, and others. Proper labeling, containers, transport media, and aseptic technique are also covered to ensure sample quality and viability.

1. SPECIMEN COLLECTION, TRANSPORT AND
ASPETIC TECHNIQUE
Mr.ManojMahato

2. INTRODUCTION
 Specimen collection is a prior procedure towards a better
diagnosis.
 Collection should be of a quality, that means; right
specimen, collected at right time and transported at a
right way to the right laboratory.
 Collection should be from a right patient.
 Handling ensures right collection and quality of specimen
as specified by SOP.

3. “SPECIMEN COLLECTION IN
MICROBIOLOGY, TO ISOLATE AND
IDENTIFY THE CAUSATIVE AGENTS
FORMS BACK BONE OF THE
INVESTIGATIVE PROCEDURES”
SPECIMEN COLLECTION IS IMPORTANT .

4. CONSIDERATION
 Start collection of specimens for all cultures before starting
an Antibiotic.
 If patient have had antibiotics, stop giving antibiotics at
least for 48-72 hrs and collect specimen.
 Avoid contamination from indigenous flora (normal flora),
whenever possible to ensure a sample representative of the
infections process.
 Collect samples from appropriate time and site with aseptic
technique.
 Collect adequate volumes; insufficient material may yield
false-negative results.
 Prompt delivery to laboratory (preferably, not later than half
an hour)

5. SPECIMEN TRANSPORT
 Within 2 hours of collection
 Containers should be leak-proof
 Separate section for paperwork
 Special preservatives or holding media
 Biohazard label

6. TRANSPORT MEDIUM
 Popularly used transport medium are:
-Amie 's Transport Medium
-Cary-Blair’s Transport Medium
-Stuart's Transport Medium
Cary-Blair All enteric organisms
Stuart All enteric organisms
Amies All enteric organisms
Buffered glycerol saline All enteric organisms except
Vibrios,Campylobacter
Alkaline peptone water Vibrios
V-R fluid Vibrios

7. CONTAINERS FOR SAMPLE COLLECTION
 Leak-proof
 Unbreakable
 Sterile and dry
-Containers should be sterilized either By moist heat or
by dry heat or by radiation but never by disinfectant
or antiseptic.

8. REJECTION CRITERIA
 Unlabeled or mislabeled specimens
 Use of improper transport medium
 Excessive transport time
 Improper temperature during transport or storage
 Improper collection site for test requested
 Specimen leakage out of transport container

9. AN IDEAL SPECIMEN FORM
 Patient’s name……………Age/ sex….
 Address …. IP/OP No …..
 Date and time of collection
 Lab number
 Ward
 specimen type
 diagnosis, and test requested.
 Nature of specimen
Doctor/Staff
…………….
 signature

10. LABEL HIGH RISK SPECIMENS
 Sputum with suspected
Tuberculosis
 Fecal samples suspected with
Cholera, Typhoid,
 Anthrax ?
 Serum when suspected with HIV/
HBV/HCV, infections

11. SELF PROTECTION
 A few ways to make sure your role in
the collection process is carried out
with efficiency, orderliness and safety

12. STANDARD PRECAUTIONS
 All specimens should be presumed to contain
transmissible agents and therefore should be collected
and handled using standard precautions.
 Use of gloves, gown, mask, and protective eyewear
when there is a risk of coming in contact with the
specimen
 In most clinical laboratories, a special area is designed
for processing clinical samples for culture

13. EQUIPMENT NEEDED
13
 Chlorhexidine swabs /Alcohol swabs
 Collection tubes
 Collection tubes
 Gloves (sterile &nonsterile)
 Tourniquet
 Sterile gauze pad
 Adhesive strip or tape
 Self-sticking patient labels
 Plastic zip lock specimen bags
 Hand towel or absorbent pad.
 Slides.

15. Label the tubes, checking the requisition for the proper identification.
15

16. SPECIMENS
 For isolation and diagnosis of microrgansm following
specimen collected inmicrobiology laboratory
 These are:
-sputum
-throat swab
-urine
-stool
-bone marrow
-rectal swab
-vaginal swab-
Cerebral spinal fluid

17. SAMPLE COLLECTION OF RESPIRATORY TRACT
 Collection of specimen in the case of RTI poses a
number of problems because , there is enormous
commensal flora that colonizes this tract.
 Therefore, the specimen collection is very crucial
and specially in case of viral infections of RT.
 One has to avoid contamination of the specimens.
 RT is broadly divided into:

18. ANATOMYOFRESPIRATORYTRACT

19. UPPER RESPIRATORY TRACT
1.Oral swab:
• Remove the oral secretions or debris
from the surface of lesion with swab and discard
• Using 2nd swab ,vigorously specimen the
lesion avoiding any areas of normal
tissue
2. Nasal swab:
• Use swab moistened with sterile
saline.
• Insert approx. 2cm into nares
• Rotate swab against nasal mucosa

20. 3. Nasopharyngeal:
A. Swabs:
• To collect nasopharyngeal cells, all mucus is removed
• Small flexible nasopharyngeal swab is inserted along the nasal
septum to the posterior pharynx
• Rotate slowly for 5 sec. against the mucosa several times
B. Aspirate :
• Is collected with a plastic tube attached to 10 ml syringe or suction
catheter
C. Washings:
• Is obtained with a rubber suction bulb by instilling and
withdrawing 3-7 ml of sterile buffer saline

21. THROAT SWAB
/ 42
 Swabs- Cotton, Darcon or Calcium alginate-
tipped swabs
 For 8 hrs before swabing, must not be treated
with antibiotic and mouth gargle
 In good light, collect as much exudate as
possible from tonsils, posterior pharyngeal wall
or other inflamed sites
 Swab- rubbed with rotation over one tonsillar
area, then arch of soft palate and uvula, the
other tonsillar area and finally posterior
pharyngeal wall
 Contamination from oral flora should be
avoided
Transport:
•Within 24hrs/RT

22. can lead to
respiratory
blockage.
Pseudomembrane
on tonsils

23. -if can’t be delivered within 1 hour, refrigerate at 4oC
-Alternatively, can be stored in tube with silica gel and
transported
-moist swab- can be cultured upto 4 hrs
-Group A streptococci- highly resistant to desiccations
(survive in dry swab for 48-72hrs)
- can be placed in glassine paper envelopes for transport

24. Specimen:
 Bronchial washing and lavage, sputum etc
 blood sample for serology, serum stored at refrigerator
until processing
Transportation: for virus detection, should be transported
immediately
 specimen should be placed in viral transport media and
transported in ice box

25. SPUTUM COLLECTION
 Proper patient instruction
 Food should not have been ingested for 1-2 h prior to
expectoration
 The mouth should be rinsed with saline or water
 Patient should breathe and cough deeply
 Patient should expectorate into a wide mouth container
 Transport container immediately to lab

26. SPUTUM SPECIMENS
-3 major types for
 Culture and sensitivity
 Acid Fast Bacilli
 3 consecutive, early am
 Cytology
 Abnormal lung cancer by cell type
 3 early am

27. SPUTUM
 Material from lower respiratory tract infection,
most commonly sent specimen for
bacteriological examination is sputum (mixture
of bronchial washing and inflammatory exudate)
coughed up into mouth and expectorated
 Bacterial infection- purulent containing green or
yellow material with mucus
 Saliva- relatively clear and watery
 Collected in clean, dry, wide mouthed, leak-
proof container, preferably early morning sample

28. INDUCED SPUTUM
 Patients who are unable to produce sputum.
 patient to breath aerosolized droplets of a solution of
15% sodium chloride and 10% glycerin for
approximately 10 minute .
 avoid the need for a more invasive procedures, such
as bronchoscopy or needle aspiration etc

29. SPUTUM COLLECTION
 May be delegated
 Cough effectively
 Mucus from bronchus
 Not Saliva
 Record
 Color
 Consistency
 Amount
 Odor
 Document date & time sent to lab.

30. LOWER RESPIRATORY TRACT
 lower respiratory tract (trachea, bronchi & lungs) –
normally free from microorganisms
 Immunocompromized- organism from throat may
invade
 Inhaled pathogens-
Mycobacterium diptheriae, Bordetella pertusis,
Mycoplasma pneumoniae, Chlamydia, Influenza
viruses etc

31. LOWER RESPIRATORY TRACT INFECTIONS
 Acute bronchitis
 chronic bronchitis
 Pneumonia:- -Typical pneumonia
-Atypical pneumonia
-Community acquired pneumonia
-Hospital acquired pneumonia
 Others
- Lobar pneumonia
- Bronchopneumonia
-Aspiration pneumonia
-Pneumonia in the immunocompromised host

32. URINARYTRACTINFECTION
 Microbial invasion of any tissue of urinary tract
from the renal cortex to urethral meatus.
 infection of prostate and epididymis is also
included.
 2nd commonest site of infection after respiratory
tract

33. CHARECTERIA FOR URINARY TRACT INFECTION
 Frequency
 Urgency
 Dysuria
 Hematuria
 Fever
 Cloudy,
 burning micturition,
 lower abdominal pain
 bactereuria
 pyuria

34. URINARYTRACTINFECTION
 CYSTITIS:
 Gram negative bacilli: Escherichia coli, Klebsiella spp,
Proteus spp, Enterobacter spp, Citrobacter spp., Providencia
spp,Morganella,Acinatobacter,Pseudomonas etc
 Gram positive cocci: Staphylococcus aureus, Coaggulase
negative staphylococci, Enterococcus faecalis etc
 PROSTATITIS:
 Gram negative bacilli: E. coli, Haemophilus influenzae
 Gram positive bacilli: Corynebacterium spp

35.  URETHRITIS:
 Gram negative bacilli: Escherichia coli
 Gram positive cocci: Streptococcus spp, Coagulase
negative staphylococci
 Gram negative cocci: Neisseria gonorrhoeae
 Others: Trichomonas vaginalis

36.  Clean catch mid-stream urine specimen
Requirements:
-Sterile, dry, wide mouth leak proof
bottle
-Early morning (midstream) first urine
sample
 Instructions:
- male patients should wash the genital organ with
clean water
-female patients should cleanse the area around
urethral opening with clean water.
-after drying the area, midstream urine is collected
URINE COLLECTION METHODS:

37.  METHOD:
-Give patient a sterile, dry and wide-necked, leak proof
container
-Instruct to collect about 20 ml of midstream urine sample with
as little contamination as possible.
-By removing the cap bottle and after discarding the initial portion
of urine, required amount of urine sample is collected.
(If possible, the first morning sample as it the most concentrated)
-Label the container with date, name, number of patient and
time of collection
-Deliver the sample with request form to lab as soon as
possible. If not possible, can be stored at 2-8oC for 24 hrs.
-Add 0.1g/10ml boric acid powder to preserve the specimen

38. REQUIREMENTS:
- clean, dry, leak proof and sufficiently large to collect
entire specimen
Collection: early morning entire urine specimen for three
successive days
can be stored at 4oC until all three urine specimen are
collected
Or
-24 hours urine specimen
-container should be of 3 liter capacity
URINE SPECIMEN WITH SUSPECTED RENAL TUBERCULOSIS

39.  Wash hands, done apron, prepare equipment. Apply alcohol
hand rub.
 Once sufficient urine has collected in the tube, wipe the
sampling port with an alcohol-impregnated swab. Allow to dry.
 Stabilising the tube below the sampling port, insert the needle
into the port at an angle of 45.
 Aspirate the required amount of urine
 Inject urine into sterile specimen container.
 Wipe the sampling port with an alcohol-impregnated swab and
allow to dry.
 Unclamp the catheter tubing as required.
 Dispose of waste, remove apron, wash hands thoroughly.
 Complete documentation.
 Dispatch the specimen to the laboratory.
CATHETERIZED URINE SPECIMENS

40. PEDIATRICSPECIMEN
1) Supra-pubic aspiration
-Ask assistant to hold infant supine
with legs extended
-Ask parent to be ready to catch urine
if the patient voids
-Wipe the skin with an alcohol swab
Insertion point:
 Midline
 Lower abdominal crease
-Insert needle perpendicular to the skin,
aspirating gently as you advance the
needle.
--Thus obtained urine ,remove needle
and squirt urine into sterile urine jar

41.  Bladder washout
 Intravenous pyelography
 Micturating cystography
 Urethrescopy
 Ureteric catheterization
 Renal biopsy
INVASIVE METHODS:

42. GENITAL TRACT INFECTION
 Genital ulceration
 Urethral discharge
 Abnormal vaginal discharge
 Asymptomatic infection

43. SEXUAL TRANSMITTED
DISEASE
Genital ulcers
 earliest clue and common manifestation
 Painless ulcers
 Treponema pallidum---------Syphilis
 Chlamydia trachomatis-----Lymhogranuloma venerum
 Calimatobacterium granulomatis--------Donovanosis
 Painful ulcers
 Haemophilus ducreyi-----------Chancroid
 Herpes simplex virus types 1 & 2 ------Herpes genitallis

44.  Genital discharge
a) Gonococcal urethritis- Neisseria gonorrhoeae
b) Non-gonococcal urethritis
- C. trachomatis
-Ureaplasma urealyticum
-Mycoplasma genitalium
-Acintobacter spps
-Bacteroides
 -Candida albicans

45.  Vaginal Discharge:-- Trichomonas vaginalis
-Candida albicans
-Gardnerella vaginalis
and other anaerobes
 Asymptomatic: - N. gonorrhoeae
-C. trachomatis
-HIV, HBV,CMV

46. COLLECTION OF UROGENITAL SPECIMEN
 Specimen required for diagnosis of gonorrhoea
 Female patients
 Smear of mucopus from cervix and urethra
 Swab of urethral discharge in Amies transport media or
inoculated directly in selective media
 Male patients
 Smear of urethral discharge
 Swab of urethral discharge in Amies transport media or
inoculated directly in selective media

47.  Collection of urethral specimen
 Patient should not have passed urine preferably for 2 hrs before
sample collection
 Cotton swab treated with charcoal, Calcium alginate (not for HSV,
gonococci, Chlamydia and mycoplasma) and Dacron tipped swab)
 Urethral swab- swab is inserted about 2 cm into urethra and
rotated gently. (mainly for chlamydia)
 Collect a sample of pus.
 Contamination from indigenous commensal flora should be
avoided
 Transport rapidly and ambient temperature conditions
 Make a smear for gram stain and process for culture

48.  Collection of cervical specimen
 Insert vaginal speculum
 Cleanse cervix with swab moistened
with sterile physiological saline
 Pass sterile cotton swab into
endocervical canal and gently rotate
 Insert swab in Amies transport medium
 Make a smear for staining

49.  Collection of vaginal specimen
 Collect vaginal discharge in sterile cotton swab
 Insert into Amies transport media
 Make smear for staining
 Wet preparation is helpful for protozoal parasites and
fungi
 Report the appearance of discharge
 Yellow green purulent- T. vaginalis
 White discharge (pH <4.5)- C. albicans
 Grey offensive (pH> 4.5)- G. vaginalis

50.  Collection of other urogenital specimens
 Syphilis
Wearing rubber gloves, cleanse area around ulcer with
physiological saline
Collect serous exudate on cover glass and invert on
glass slide
Transfer immediately for DGI

51. GASTROINTESTINAL INFECTION
 Microorganism that cause GI infections
 Invasion- Shigella spp., EIEC, E. histolytica, B. coli,
Yersinia spp etc
 Toxin production
 Enterotoxin- V. cholerae, Shigella dysenteriae type 1, ETEC,
Salmonella, Aeromonas spp, Clostridium spp etc
 Cytotoxin- Shigella spp, EHEC etc
 Neurotoxin- C. botulinum, S. aureus, B. cereus etc

52. COLLECTION OF GIT TRACT SPECIMEN
 Faeces for microbiological examination should be collected during
acute stage, preferably before antimicrobial treatment
 Fresh specimen is preferred to rectal swab and faecal swab
Rectal swab: if not possible to obtain stool sample
 Collection of stool sample
 2 small wooden stick and suitable clean, dry, disinfectant free bedpan or wide-
necked, leak proof container
 Specimen should contain at least 5 g of faeces
 Sample containing blood, mucus or pus is better
 Shouldn’t contaminated with urine
 Should be transferred to lab within 2 hrs
 If not possible, a small amount of faecal specimen (with mucus, blood and
epithelial if present) should be collected 2-3 swabs
 Transport in Cary-Blair, Stuart or Amies media

53.  COLLECTION OF RECTAL SWAB
 Moisten a cotton swab with sterile water
 Insert swab through rectal sphincter, rotate and withdraw
 Examine swab for faecal staining
 Place swab in sterile tube containing cotton lug or screw-
cap if processed within 2 hrs
 If need to kept for longer period- place in transport
medium

54. WOUND INFECTIONS
 Wound infection can occur as a
complication of surgery, trauma, bites or
diseases
 Postoperative infections:
 Infection from patient’s own normal
flora or organism present in
environment
 Depends on site of wound eg.
Appendectomy or lower GIT surgery
– E. coli, Streptococci, Bacteroides,
Clostridium and other anaerobes

55.  Collection of specimen
 Best collected at time of
incission or drainage
 Special care to avoid
contamination from skin
commensals
swab: collect sample aseptically
and transfer to amies transport
media or sterile container
Aspirates: discharging pus and
granules (if present) in sterile
leak proof container

56. DEFINITION
 Bacteremia - presence of viable
bacteria in the blood stream
 sepsis- bacteremia associated with
an inflammatory response from
the body (systemic inflammatory
response syndrome- rapid
breathing , low blood pressure and
fever etc.)
 Microorganism present in
circulating blood- continuously,
intermittently or transiently
 serious immediate consequences-
shock, multiple organ failure,
DIC, and death.

57. Bacteria commonly isolated from blood cultures
 Salmonella spp
 Escherichia spp
 Klebsiella spp
 Enterobacter spp
 Proteus spp
 Pseudomonas spp
 Acinatobacter spp
 Anaerobic bacteria such as
Bacteroides and Clostridium spp
 Coagulase negative staphylococci
 Staphylococcus aureus
 Viridans streptococci
 Enterococci spp
 Beta- hemolytic streptococci
 Streptococcus pneumoniae

58. Organisms commonly associated with bloodstream
invasion from extravascular sites of infection
Organism Extravascular site of infection
Haemophilus influenzae type b Meningitis, epiglotitis, periorbital
region
Streptococcus pneumoniae Meningitis, sometimes lung
Neisseria meningitidis meninges
Brucella spp Reticuloendothelial system
Salmonella typhi Small intestine, lymphnodes of
intestine, RE system
Listeria meninges

59. CLINICALMANIFESTATIONS
 Septicemia: bacteria and their products causing harm to host
: signs and symptoms of septicemia
-fever
-hypothermia
-chills
-hyperventilation and subsequent respiratory alkalosis
-skin lesions
-change in mental status and diarrhea
Septic shock syndrome -fever, acute respiratory distress, renal
failure, intravascular coagulation, and tissue destruction

60. DETECTION OF BACTEREMIA
 Specimen collection
1) Preparation of site:
first vein is chosen
-if patient has an existing IV line, blood
should be drawn bellow existing line
Antisepsis: 70% alcohol and tincture of
iodine
2) Method:
-choose the vein to be drawn
-using 70% alcohol, cleanse approxm 5cm in
dia
-apply 2% tincture of iodine(or povidine
iodine)
or 1 to 2% chlorhexidine in ever widening
circle
-allow to dry the skin for at least 1 minute
-insert the needle into the vein and withdraw
blood

61.  Specimen volume
i) adults: low number of CFU
-collection of 10 to 20 ml of blood per culture
-increase yield of 3.2% for each ml of blood cultured.
ii) Children: for infants and children, only 1 to 5 ml of blood is
drawn
-high levels of bacteremia in some infants

62. NUMBER OF BLOOD CULTURES
 If the volume adequate: 2-3 blood culture is sufficient
 Endocarditis:
not received anyibiotics- a single blood culture pos. 90-95%
-second blood culture at least 98%
received prior antibiotics therapy- 3 blood collections of 10-20 ml each an
additional blood culture or 2 on second day.
 Bacteremic pts- 80 to 92 % by 1st blood culture
-90 to 99% by 2nd blood culture
-99.6% by 3rd blood culture

63. ANTICOAGULATION:
-Blood drawn for culture must not be clotted
-Heparin, EDTA and citrate inhibit bacterial growth, not used
-Sodium polyanethol sulfonate : 0.025 to 0.03%
DILUTION:
-largest feasible vol. of blood (10ml) with the smallest amount of media
-still encourage the growth of bacteria and dilute antibacterial
components of blood

64. BLOODCULTUREMEDIA:
-Diversity of bacteria- equally various types of culture
media
-commonly used:- nutrient broth
- trypticase soya broth
- BHI broth
- supplimented peptone water
- thioglycolate broth etc
ADDITIVES:
-cellwall deficient bacteria need osmotic stabilizers eg.
sucrose, mannitol or sorbose
-penicillinase to inactivate penicillin
-recent years, use of resins to inactivate antibiotics

65. FLUIDS
 CSF
 Pleural fluid
 Peritoneal fluid
 Joint fluid

66. COLLECTIONANDTRANSPORT
 After aspiration, aseptically dispense
 2-3ml into dry, sterile and screw-cap tube or
bottle
 9ml into screw cap tube or bottle with 1 ml tri-
sodium citrate 3% w/v and mix well.
 Label and deliver to lab as soon as possible

67.  Laboratory diagnosis
 Specimen obtained after careful preparation of skin site
 Anaerobes survive long time in tissue
 A small amount of non-bacteriostatic saline can be added to
keep specimen moist
 Legionella spp. may be inhibited by saline
 Formaldehyde fixed tissue is not suitable for recovery of
organism
 Grinding may destroy some organism including fungal cells . So
mince the larger tissue to culture
 As surgical specimen are obtained at great risk and expense,
save a portion of original tissue in refrigerator or at -70oC

68.  Sample collection:
 usually performed by medical officer
 Bone marrow aspiration
 For diagnosis of certain diseases eg. brucellosis,
histoplasmosis, blastomycosis, tuberculosis and
leishmaniasis
 Bone biopsy
 Small piece of infected bone- for diagnosis of
etiologic agent of osteomyelitis

69. CEREBROSPINAL FLUID
Procedure: Lumbar Puncture Pt lies in L
lateral decub position, knees to chest
 Aim for the L3-L4 or L4-L5
intervertebral space
 Posterior iliac crest as marker for
L4-L5 space
 Prep/drape lower back in sterile
fashion...lidocaine
 Insert LP needle pointing towards
umbilicus with the bevel up
 Obtain opening pressure

70.  It is important when
there is delay in
transportation of
specimens to Laboratory
do not keep in
Refrigerator, which tends
to kill H. Influenza
 If delay is anticipated
 leave at Room
Temperature.
PRESERVATION OF CSF:

71. THANK YOU….