CHAPTER I

COLLECTION, TRANSPORT AND

EXAMINATION OF BACTERIOLOGICAL
SPECIMENS
 ACKNOWLEDGMENT
• ADDIS ABABA UNIVERSITY
• JIMMA UNIVERSITY
• HAWASSA UNIVERSITY
• HARAMAYA UNIVERSITY
• UNIVERSITY OF GONDAR
• AMERICAN SOCIETY OF CLINICAL PATHOLOGY
• CDC- Ethiopia
 Objectives
Upon completion of this unit of instruction, the student will
be able to:
• Describe types of specimens used for bacterial analysis;
specimen collection requirements, transportation &
storage; and examination of clinical specimens
• List the medically-important species of bacteria
• Recognize diseases caused by medically important
bacterial species
 Objectives
• Describe the virulent factor of pathogenic species
• Discuss pathogenicity, clinical manifestations, laboratory
diagnosis, prevention & control of members of the
pathogenic bacterial species
• Perform laboratory quality control in bacteriology
 Introduction to specimen collection
 Definition of specimen
o Specimen is a part taken to determine the character of the whole
 Safety during collection, processing & transportation
o Masks (e.g. sputum)
o Gloves
o Protective eye ware (goggles)
o Protective clothing (gown)
 Specimen collection
o Correct treatment depend on accurate result
o Accurate result depend on quality specimen
o Quality specimen depend on proper :
 Collection
 Transportation
 Processing of specimens
o Purpose:
 provide high yield positive result
Provide that a negative result indicates the absence of the
pathogen
 Introduction ....
 Importance of Quality Specimen on Patient Care
o Key to accurate laboratory diagnosis

o Directly affects patient care & patient outcome

o Influences therapeutic decisions

o Impacts hospital infection control

o Impacts patient length of stay, hospital costs, and laboratory

costs

o Influences laboratory efficiency

 Basic Concept in Specimen Collection

If pathogens are to be isolated successfully the following points must be

considered:

1. Site selection
a. Clinician
 Should locate right anatomic site & select appropriate tests &
specimens based on:
 Physical examination (sign & symptoms)
 Radiological examination
b. Laboratory personnel
o Should collect specimens from actual infection site with little
external contamination by using :
a. Aseptic technique
• To prevent contamination of specimen &
• To protect the patient from infection
a. Sterile container
o Should collect specimens from right site
 Sites of Infection where the Specimen is Likely to
Become Contaminated During Collection
o Lower respiratory tract ----- Oro-pharynx
o Bladder ……………..… Urethra
o Cervix …………. Vagina
 Approaches to Avoid contamination :
o Careful patient education
 There are occasions when patients participate actively in

specimen collection (e.g. sputum, urine)

 Therefore, they must be given full instructions &
cooperation
by the care giver
o Educating the clinicians
 How to collect & transport specimens through written
document & make available at every patient care unit
 2. Volume of specimens
 Collecting & processing too little specimen lower sensitivity
 Collecting adequate volume:
o enhance recovery of the pathogen
o enable to perform all procedures required or to permit
complete examination
 For example;
o Sputum: 5 -10 ml for mycobacterium examination

o Blood:
 Serology: minimum 4 - 5 ml
 Culture: 10 – 20 ml (adult) & 1-5ml (infant)
o CSF: 5 – 10 ml
 3. Time of collection
 Provide best chance of recovery of the causative agent
 Before collecting a specimen for bacteriological examination,
the right time at which the organism proliferate maximum
should be take in to account and before the pt take
antimicrobial drug
o Sputum & urine
 early in the morning soon after the patient awaken

o Blood
 when the patients temperature begins to rise
4. Age of specimens
o Age of the specimen directly influences the recovery of
Microorganisms

5. Stage of the disease at which the specimen is collected

o Enteric pathogens are present in great numbers during the
acute or diarrheal stage of intestinal infection
 7. Labeling
Make sure that you are collecting/drawing the right person first.
 Then label with:
o Patient name
o Unique identification number
o Patient demographic information
o Specimen collection date
o Specimen collection location
o Diagnostic test results
 Labeling ...
 During Labeling:
o Make sure that container label & the requisition match
o Label should be on the container not on the lid, since the
lid can be mistakenly placed on a different container
o Ensure the labels on the containers are adherent under
refrigerated conditions
Collection techniques:
• the laboratory should issue written instructions to all those
responsible for collecting specimens including the staff of
wards, out patient clinics, and health centers .
• The following precautions apply to the collection of most
microbiological specimen;-
A. Use a collection technique that will ensure a specimen
contains only those organism from the site where it was
collected
- A strictly sterile (aseptic) procedure is essential when collecting
from sites that are normally sterile (Ex blood, CSF)
- Avoid contaminating discharges or ulcer material with skin
commensals
- The swab used to collect the specimen must be sterile and free
from any antibacterial substances
Collection techniques….
• Collect specimens in sterile, leak- proof, dry containers, free
from all
traces of disinfectant
• Containers must be clean but need not be sterile for the
collection of sputum and stools
• Containers must be easy for patients to use
• Patients must be instructed how to collect aseptically
• Macroscopic feature of the sample must be reported
- Color
- Presence of pus, blood, mucus or parasites
- The appearance of urine, pus, vaginal
discharge , faeces, CSF
 B. Each specimen must be accompanied by a request form
which gives:-
- the patient’s name, Age
- Patient’s number, ward or health center
- type of specimen and the date and time of its
collection
- Investigation requested
- Whether the patient has started any antimicrobial treatment
Specimens containing dangerous pathogens
• Those delivering, receiving, and examining specimens must be
informed if a specimen is likely to contain highly infectious
organisms.
• Such a specimen should be labeled HIGH RISK and when ever
possible carry a warning symbol such as a red dot or star,
which helps for easy recognition
C. Preservatives and transport media for microbiological
specimen
• In general, specimens for microbiological investigations should
be delivered to the laboratory as soon as possible. This will
help to ensure that pathogens are living when they reach the
laboratory
• If there is any delay use chemical preservative. This will help to
prevent organisms from dying due to

- Enzyme action
- Change of PH
- Lack of essential nutrients
- A transport medium is usually needed to preserve an aerobes
- Some of the transport mediums are
 Amies transport medium- for specimens collected on swabs
(especially N. gonorrhoea)
 Cary- Blair medium- for faeces specimen (especially which
contains salmonella, shigella, campylobacter or vibrio species
- Same of chemical preservatives are
 Boric acid  added in urine
 Cetylpyridium chloride sodium chloride (CPC-Nacl) added to
sputum
Note - chemical preservatives which contain formaldehyde should
not be used for microbiological samples because formaldehyde
with kill the bacterias
- Refrigeration at 4-100C can help to preserve cells and
reduce the multiplication of commensals in unpreserved
specimen
1. COLLECTION, TRANSPORT AND EXAMINATION OF
SPUTUM
Collection and transportation of sputum In hospital
Microbiology Lab.

• Sputum for microbiological investigation is collected and

transported as follows:-
– Give the patient a clean (need not be sterile), dry, wide-
necked, leak proof container, and request him to cough
deeply to produce a sputum specimen
Important :- the specimen must be sputum, not saliva. sputum is
best collected in the morning soon after the patient wakes and
before and before any mouth wash.
- If the patient is young child and it is not possible to obtain
sputum gastric washings can be used for the isolation of M.
tuberculosis
• Label the container

• If pneumonia or bronchopneumonia is suspected, deliver the

sputum to the Lab with a little delay as possible, because H.
influenzae and S. pneumoniae require culturing as soon as
possible
• If the specimen is for the isolation of M. tuberculosis, it should
be delivered to the Lab with in 2 hours or kept at 40C
Note:- specimens for the isolation of S. pneumonia and H.
influenzae must never be refrigerated
- if pneumonic plague is suspected : Deliver the sputum to
the Lab. As soon as possible. Make sure the specimen is
marked HIGH RISK
In a health center for dispatch to a microbiology laboratory
Collect the sputum in a container supplied by the Microbiology lab
 If pneumonia and broncho pneumonia pathogens suspected:-
- Collect a purulent part of the sputum on a cotton wool swab
and insert in a container of Amies transport medium
- Label the container using a lead pencil
- Make a smear of a purulent part of the sputum on a slide for Gram
stain
- Fix using heat or alcohol
- Send the swab and smear with a request form to reach the
microbiology Lab with in 6 hrs
 If pneumonic plague is suspected:
- Send a swab of the sputum in cary- Blair transport medium to the
Microbiology Lab with in 4-6 hrs
 If M. tuberculosis is suspected
- Make a smear of the sputum on a slide for AFB staining
using
caseous particles and the most purulent material
- Mix the sputum in its container with equal volume of CPC-
Nacl
solution
Note: CPC-Nacl solution digests (liquefies) sputum in about 24
hours and prevents the growth of other bacteria M. tubercluosis
will remain viable in the sample for up to 8 days
- Send the smear and CPC-Nacl treated sputum to
microbiology lab. With in one week
Monica Cheesbrough, District LaboratoryPractice in Tropical Countries Part 2. 2nd ed. 2006.
2. COLLECTION, TRANSPORT, & EXAMINATION OF THROAT
AND MOUTH SPECIMEN
 In a hospital with a microbiology laboratory
• In a good light and using the handle of a spoon to depress the
tongue, examine the inside of the mouth for inflammation, and
the presence of any membrane, exudates, or pus.
– With diphtheria, a grayish- yellow membrane (later becoming
grayish green black & smelly)
– With C. albicans, patches of white exudates may be seen
attached in places to the mucous membrane of the mouth
etc
• swab the affected area using a sterile cotton or wool
swab. Then return it to its sterile container
• With in two hours of collection, deliver the swab with a
request form to the laboratory.
 In a health center for dispatch to a Microbiology
Laboratory
– Using a sterile swab (supplied in a tube of silica gel).
Collect a specimen from the infected area.
– Send the swab with its request form to reach the
microbiology Lab with in 3days
Monica Cheesbrough, District LaboratoryPractice in Tropical Countries Part 2. 2nd ed. 2006.
3. Collection and Transport of upper respiratory track
specimens
 In hospital with a microbiology laboratory
• Nasopharyngeal aspirates;-
 Gently pass a sterile catheter through one nostril as for the
nasopharynx
 Attach a sterile syringe to the catheter, and aspirate aspecimen
of mucopus
 Dispense the specimen into a small sterile container
 Label, and deliver with a complete request form to the
laboratory
• Pernasal swabs for the culture of B. pertussis:-
 Using a sterile cotton wool swab attached to an easily bent
piece of wire, gently pass the swab along the floor of one
nostrile directing the swab down wards and back wards as far
as the nasopharynx
 Taking care not to contaminate the swab, replace it in its
sterile container
 Label, and deliver immediately to the laboratory with a
request form
• Anterior nasal swabs to detect carriers:-
 Using a sterile cotton wool swab moistened with sterile peptone
water, gently swab the inside surface of the nose
 Taking care not to contaminate the swab, replace it in its sterile
container
 Label, and with in 2 hrs deliver the swab with a request form to
the laboratory
 In a health center for dispatch to a microbiology laboratory
• Nasopharyngeal aspirates:-
 Collect in the same way as collected in hospital and make a
smear and dispatch as described for sputum
• Pernasal swabs for culture of [Link]
 Swab in the same way as described for hospital
 Insert the swab in a bijou containing special Bordetella
transport medium and seal the cap with adhesive tape.
 Send the swab to the microbiology laboratory to reach with
in 2 days
 Laboratory examination of upper respiratory track
specimens
day -1
- culture the specimen
* Blood agar and chocolate agar
 to detect [Link], [Link] and [Link] carriers:-
– Inoculate the swab on chocolate agar
– Incubate the plate in CO2 enriched atmosphere at 35-
370C/48hrs
– Examine for growth after overnight incubation
*Charcoal cephalexin blood agar if whooping cough is
suspected
• Inoculate the swab over the entire surface of a plate of
charcoal cephalexin blood agar
• Incubate the plate aerobically in a moist atmosphere in a
plastic bag or polythene bag or polythene container with a
wet piece of cotton wool ) at 35-370C 16 days,
• Examine for growth after about 48 hrs incubation
Culture of a swab received in Bordetella transport
Medium:-
◦ Inoculate the swab on a plate of CCBA as described
above
◦ Return the swab to its container and incubate it at 35-
370
◦ Incubate the plate as described above
◦ Examine the plate after 48 hrs incubation
◦ If no growth Is seen, inoculate a second plate of CCBA
with the incubated swab
◦ Re incubate the first plate for a further four nights
◦ Examining for growth every 24 hrs. Incubate the second
plate up to 6 days
 * Day- 2 and onwards
II. Examine and report the cultures
*Blood agar and chocolate agar cultures
Look especially for colonies that could be:
 [Link]
 [Link]
 [Link]
 [Link]
4. COLLECTION, TRANSPORT AND EXAMINATION OF EAR
DISCHARGES
1 collection and Transport of Ear discharges
 In a hospital with a microbiology laboratory
• When ever possible, collect or aspirate a small amount of the
discharge in a sterile leak proof container
• Label and send to the laboratory as soon as possible with the
request form
• If an actual specimen of the discharge can not be obtained,
collect, a specimen on a sterile , dry, cotton wool swab
• If fungal infection is suspected do KOH test from the sample
• Deliver the swab and the sample as soon as possible
 In a health center for dispatch to a microbiology Laboratory.
• collect a specimen of the discharge on a sterile cotton wool
swab
• Place the swab in Amies transport media and tight the bottle
clap
• Make a smear of the discharge on a slide (for Gram stain)
• Label and send the specimens to reach microbiology
laboratory with in 6 hrs
2 Laboratory Examination of Ear discharges
* Day- 1
I. Culture the specimen
◦ Blood agar and MacConkey agar
 Inoculate the sample on Blood agar and MacConkey agar
 Incubate both plates aerobically at 35-870C over night
◦ Chocolate agar if the patient is a child
 Inoculate the specimen on chocolate agar for the isolation of
[Link]
 Incubate it in a CO2 enriched atmosphere at 35-370C/48 hrs
 Examine for growth after overnight incubation
◦ Blood agar (kanamycin) for anaerobic incubation if infection is
chronic
 Inoculate the specimen on blood agar, preferably that which
contain kanamycin
 Incubate the plate anaerobically for up to 48hrs
 Check for growth after over night incubation
◦ Sabouraud agar if a fungal infection is suspected
 Inoculate the sample and incubate it at room temperature
for up to 6 days
◦ Gram smear
 Do gram stain of the smear and examine for pus cells and
bacteria and fungi
 G+ve, cocci that could be [Link]
 G+ve, streptococci or diplococci that could be pathogenic
streptococci
 G-ve, rods that could be ,
 [Link]
 [Link]
 [Link] spp.
 Proteus spp.
 [Link]
 G+ve yeast cells that could be candid spp.
◦ KOH test if fungal infection is suspected
 Mix a small amount of the specimen with a drop of KOH
(20%w/v) on a slide, and cover with a cover glass
 After 10 minute examine the preparation microscopically
using 10x and 40x objectives
III. Examine and report the cultures
• On day-2
 Blood agar and MacConkey agar cultures
◦ Look for colonies that could be:-
 [Link]
 [Link]
 [Link]
 [Link]
 [Link]
 Proteus spp.
◦ Chocolate agar culture
 look for colonies that could be [Link]
 Anaerobic blood agar (kanamycin) culture
 Look for colonies that could be bacteriodes spp. In
Gram stained smear the organisms appear as pale
stained, small Grame-ve rods
◦ Sabouraued agar culture
 Look for colonies which could be candida spp
 5. COLLECTION, TRANSPORT, AND EXAMINATION OF EYE
SPECIMENS
1 Collection and Transport of eye specimens
 In a hospital with a microbiology Lab
• specimens from the eye must be cultured as soon as possible
after collection, because the natural secretions of the eye
contain anti. Bacterial enzyme
• Using a dry sterile cotton wool swab, collect a specimen of the
discharge
• Inoculate the discharge on the following media-
• Blood agar
• Chocolate agar
• MNYC
Make a smear for a gram stain
If [Link] suspected make another smear for Giemsa
stain
Label both the culture plate and the slides
As soon as possible deliver to microbiology Laboratory
 In a health center for dispatch to a microbiology Laboratory
Using a sterile cotton wool swab, collect a specimen of the
discharge and insert in Amies transport media
Make also a smear of the discharge on a slide
Send the specimens with a request form to reach the
microbiology Laboratory with in 6 hrs.
2. Laboratory examination of eye specimens
* day - 1
I. Culture the specimen
Blood agar and chocolate agar
 Inoculate the eye discharge on blood agar and
chocolate agar
 Incubate the blood agar plate aerobically at 35-370C
overnight
 Incubate the chocolate agar plate in a CO2 enriched
atmosphere for 48hrs. checking the growth after over
night incubation
MNYC selective medium if gonococcal conjunctivitis is
suspected
 Inoculate the eye discharge on a plate of MNYC medium
 Incubate at 35-370C overnight
 Incubate the chocolate agar plate in a CO2 enriched
atmosphere for 48 hrs. checking the growth after over night
incubation
MNYC selective medium if gonococcal conjunctivitis is
suspected
 Incubate the eye discharge on a plate of MNYC medium
 Incubate at 35-370C in a CO2 enriched atmosphere over
night
II. Examine the specimen microscopically
Do Gram stain and examine the smear using the 40x
and 100x objective for pus cells and bacteria
Look especially- G-ve diplococci intracellular bacteria
that could be [Link]
G+ve streptococci or diplococci, that could be
Haemophilus SPP.
Giemsa smear if [Link] is suspected
◦ After staining the fixed smear, examing using 40x and
100x objectives
◦ Look for epithelial cells that contain inclusion bodies
◦ Report the smear as’chlamydial inclusion bodies
present’ or ‘No chlamydial inclusion bodies seen’.
III. Examine and report the cultures
On day 2 and on wards
• Blood agar and chocolate agar colonies
• Look especially for colonies that could be:-
• [Link]
• [Link]
• Beta hemolytic streptococci
• [Link]
• [Link]
• MNYC medium culture
• [Link] grows rapidly on MNYC selective medium
6. COLLECTION, TRANSPORT AND EXAMINATION OF SKIN
SPECIMENS
1 Collection of skin specimens and ulcer material
 In a hospital with a microbiology laboratory
• Using a sterile dry cotton wool swab, collect a sample of
discharge from infected tissue
• If there is no discharge, use a swab, moistened with
sterile physiological saline
• Insert the swab in a sterile tube
• If the tissue is deeply ulcerated and necrotic (Full of dead
cells):-
• aspirate a sample of infected material from the side
wall of the ulcer using needle and syringe
• Fluid from pustules and blister:-
• Aspirate a specimen using a sterile needle and
syringe

 Serous fluid from skin ulcers, papillomas, or papules, that

may contain treponemes:-
◦ Collect a drop of the exudates directly on a clean cover
glass and invert on a clean slide
◦ Deliver immediately the specimen to the Lab. For
examination by dark- field microscopy
◦ As soon as possible deliver the specimen to the Lab
◦ If the specimen has been aspirated, transport the needle
and syringe in a sealed water proof container
Skin specimen for ring worm fungi should be collected and
examined by KOH technique.
 In a health center for dispatch to a microbiology lab.
 Collect the specimen using a sterile cotton wool swab
 Inset in a container of Amies transport medium
 If the materials is aspirated fluid from a pustule transfer the
fluid to a sterile, leak- proof container
 Stopper, and seal in a leak- proof plastic or metal container
 Make a smear of the material on a clean slid of allow air dry
 Label the specimens
 Send the specimens to reach microbiology Lab with in 6 hrs
2 Lab. Examination of skin specimens
* Day-1
I-Culture the specimen
◦ Blood agar and MacConkey agar
 Inoculate the specimens on these media
 Incubate at 35-370C over night (aerobically) for the pus
from wounds culture anaerobically
 Modified Tinsdale medium (MTM) if cutaneous diphtheria is
suspected
 Inoculate the specimen on MTM to isolate c. ulcerance
 Incubate the plate aerobically at 35-370C /46hrs
 Examine for growth after over night incubation
 Blood agar and MacConky agar at room temperature if
bubonic plague is suspected
 Inculate the specimens on these media
 Incubate aerbically at room temperature for 48hrs.
 Examine for growth after overnight incubation
– Lowen stein Jensen medium if Buruli ulcer is suspected
• Make two smear of the specimen
• Then decontaminate the swab by immersing it in 4% NaoH
solution for 10 minute
• Inoculate the decontaminated specimen on two slops of LJ
media
• Incubate one slop at 35-370C as for [Link]
• And incubate the other slop at 320C for up to 8 weeks
II. Examine the specimen microscopically
• Do gram stain of the smear and examine for:-
• G+ve coca  [Link]
• G+ve streptococci [Link]
• G-ve Rods [Link], proteus spp, [Link] or other
 if cutaneous anthrax is suspected- Look for large gram variable
rods lying in chain that could be B. anthrax
 if bubonic plague is suspected, look for Gram- ve coccobacilli
that could be Y. pestis
• Giemsa or wayson’s stained smear if bubonic plague is
suspected
• Make an evenly smear and Let it air dry
• Fix it
• Stain with Giemsa stain or by the rapid wayson
technique
• Examine for bipolar staining of [Link]
• Polychrome Loeffler methylene blue smear if cutaneous
anthrax is suspected
• Make a smear and Fix it after it air dried
• And cover it with potassium permanganate (40g/l)
soln. For 10 minute
• Examine the smear under 100x objective
Examine by dark- field Microscopy to detect treponemes
 Examination of a specimen by dark field microscopy for
motile treponemes if T. pertenue and T. carateum is
suspected
Examine and report the cultures
On day 2 and on wards
Blood agar and Macconky agar culture
Look especially for colonies that could be:-
 [Link]
 [Link]
 [Link]
 Proteus spp
 [Link]
if cutaneous anthrax is suspected
 Look for large grey- white irregular colonies with wary
edges on blood agar that could be [Link]
• Modified Tinsdale medium culture
• To identify [Link]
• Blood agar and MacConkey agar at room temperature
• To look for [Link]
• LJ culture
• To look for [Link]
 7. COLLECTION, TRANSPORT, AND EXAMINATION OF PUS
FROM WOUNDS, ABSCESSES, BURNS, AND SINUSES
1. Collection of pus from wounds, Abscesses, Burns and sinuses
◦ pus from an abscesses is best collected at time the abscess
is incised and drained or after it has ruptured naturally
 In a hospital with a microbiology Laboratory
◦ using a sterile technique, aspirate or collect from a drainage
tube up to 5 ml pus
◦ Transfer to a leak proof sterile container
◦ If pus is not being discharge, use a sterile cotton wool swab
to collect a sample from the infected site
◦ Transport with Amies transport media
◦ Lable the specimen and as soon as possible deliver to the lab
In a health center for dispatch to a microbiology Lab.
• Collect a sample of the pus on a sterile cotton wool
swab
• Transport it with Amies transport medium
• If tuberculosis is suspected: aspirate a sample of
the pus and transfer it to a sterile leak- proof
container
• Make a smear for Gram stain
• Label the specimens and send with a request form
to reach the lab with in 6 hrs
2 Lab. Examination
* Day- 1
I. Describe the appearance of the specimen
 Report the colour of the pus and whether it contain granules
– Granules:- are colonies of organisms white, yellow, brown,
red or black granules of varying size, shape , and
consistency may be found in pus draining from sinuses in
mycetoma and in actinomycosis
– Pus from an amoebic abscess:- This has an acid reaction
(PH 5.2-6.7) and is usually red- brown or grey- yellow in
colour
II. Culture the specimen
* Blood agar, neomycin blood agar, MacConky agar, and
cooked meat medium
◦ Inoculate the specimen in a pair on each medium of
the above
◦ Incubate one blood agar plate and MacConky agar
plate aerobically at 35-370C over night
◦ Inucbate the neomycin plate and the second blood
agar plate anaerobically at 35-370C for up to 48 hrs
◦ Incubate the cooked meat medium at 35-370C for up
to 72 hrs. subculture at 24 hrs, and if indicated at 48hr
and 72 hr.
• LJ medium if tuberculosis is suspected
– Wait until the next morning to see whether there is growth on
the blood agar and MacConkey agar plates
– If sterile, inoculate the specimen directly on a slope of LJ
medium
– If there is growth on the blood agar and macConkey agar
plates decontaminate the specimen by 4% w/v NaoH for 10
minute
– Then inoculate on LJ medium
– Incubate the LJ medium as described for sputum
III. Examination of the specimen microscopically
– Gram stain
• Do Gram stain for a smear made from the specimen
• And examine for the following bacterias:-
 G+ve cocci - s aureus or [Link], anaerobic
streptococci or
enterococci
 G-ve Rods - Proteus spp, [Link], [Link] or
Bacteriodes
 G+ve larg rods with straight ends--- C. perfringens
• AFB smear if tuberculosis is suspected
• do AFS stain and Examine the smear for aicid fast
bacilli.
• KOH preparation if fungal or actinomycete infection is
suspected
• Follow different procedure for granulated pus
• Examine the preparation under 10x and 40 x obj
IV. Examine and report the cultures
Blood agar, neomycin blood agar, and MacConkey agar cultures
– Look for colonies especially for those which could be :-
» [Link]
» [Link]
» C. perfringens
» P. aeruginosa
» Proteus spp.
» [Link]
» Enterococci
» Bacteriod spp.
» Anaerobic cocci
• Cooked meat culture
• look for the grow of [Link] and bacterioides
spp.
• Then do gram stain of the culture
• LJ- culture
• here look for growth of [Link]
 8. COLLECTIONS, TRANSPORT, AND EXAMINATION OF
UROGENITAL SPECIMENS
1 Collection and transport of urogenital specimens
 Collection of urethral specimens:-
• cleans round the urethral opening using a swab
moistened with sterile physiological saline
• Gently massage the urethra from above down wards, and
collect a sample of pus on a sterile cotton wool swab
• Insert the swab in a container of Amies transport medium
Note:- The patient should not have passed urine preferably for 2
hrs before the specimen is collected
• Make a smear of the discharge on a slide for staining by the
Gram technique(the smear should be made by rolling the swab
on the slide and fixed with methanol) & Label the specimens
 Collection of cervical specimens
• Moisten a vaginal speculum with sterile warm water and
insert in to the vagina
• Cleanse the cervix using a swab moistened with
physiological saline
• Pass a sterile cotton wool swab in to the endocervical
cannal and gently rotate the swab to obtain a specimen
• Insert the swab in a container of Amies transport medium
• Make a smear of the cervical mucopus on a slide for
Gram staining & Label the specimens
 Collection of vaginal specimens
• Collect a sample of vaginal discharge on a sterile cotton
wool swab and insert it in amies traport medium
• Make a smear of the discharge on a slide for staining by
Gram technique
• Label the specimens
• Appearance of vaginal discharge in infection of :-
• T. vaginalis:- yellow- green purulent with PH > 4.5
• [Link]:- white discharge with PH below 4.5
• [Link]:- Grey, offensive, thin discharge with PH> 4.5
• The normal PH of vaginal discharge (puberty to menopanse)
3.0-3.5.
 Collection of other urogenital specimens
• [Link]:
• wearing rubber gloves, cleans around ulcer
(chancre)using a swab moistened in physiolocial saline
• Collect a sample of serous exudates on a cover glass and
invert it on a slide
• Deliver immediately the preparation to the Laboratory for
examination
• H. ducreyi:
– After cleaning the ulcerated area as described in the
previous text, make a smear from the lesion for staining by
the Gram method
2 Laboratory examination of the urogenital specimens
* Day-1
I. Culture the specimen
• * MNYC medium
• Inoculate the specimen on MNYC selective medium,
prewarmed to room temperature
• With a little delay as possible, incubate the inoculated
MNYC plate in amoist CO2 enriched atmosphere at 35-
370C over night
• Blood agar (aerobic and anaerobic), MacConkey agar, and
cooked meat medium if sepsis abortion is suspected
• Inocualte the specimen on two plates of blood agar
• And incubate one anaerobically and the other aerobically
at 35-370C overnight
• Inoculate the specimen in cooked meat medium and
incubate at35-370C sub culturing as indicated at 24 hr, 48
hr, and 72 hr.
• Serum culture if chancroid is suspected
• Sabouraud medium if vaginal candidiasis is suspected
• If vaginal candidasis is suspected and yeast cells are not
detected microscopically, inoculate the specimen on
sabouraud agar
• Incubate the plate aerobically at 35-370C for up to 48 hrs.
checking for growth after overnight incubation.
II. Examine the specimen Microscopically
* Gram smear
• Do gram stain preparation and examine under 40x and 100x
obj for pus cells and bacteria
 smear from a patient with suspected gonorrhoeae
• Look for pus cells containing G-ve diplococci 
[Link]
 Smear from a patient with vaginitis
• Look especially for:- Larg G+ve yeast cells [Link]
• Epithelial cells with attached Gram variable coccobacilli
 [Link] with few or no pus cells
 Smear from septically aborted women
 Look escpically among pus cells For:-
 Large G+ve rods with steright ends  [Link]
 G+ve [Link] [Link]
 G+ve cocci [Link]
 G-ve Rods  Bacteriodes spp.
 Smear from a patient with suspected chancroid
 Look for G-ve coccobacilli showing bipolar staining
 Saline preparation [Link] (often found in chain)
III. Examine and report the cultures
* On day 2 and on wards
MNYC medium culture
 Look for colonies which look like gonorrhoeal’s colony
 Do gram stain from the colonies
 Look for Beta- lactamase production
Blood agar and MacConkey agar cultures
Look for colonies that could be:-
 [Link]
 [Link]
 C. perpringens
 Proteus spp
 Enterococci
 [Link]
• Sabouraud agar culture
• Look for the appearance of [Link] colonies
• Serum culture
• Examine a Gram stained smear from serum culture to
look for [Link]
9. COLLECTION, TRANSPORT, AND EXAMINATION OF
FAECES
1. Collection and Transport of faeces
• Faeces for microbiological examination should be collected
during the acute stage of diarrhea
 In hospital with a microbiology laboratory
– Give the patient a clean, dry, disinfectant- free bed pan or
suitable wide- necked container
– The container need not to be sterile
– ASK the patient to avoid contaminating with urine
– Transfer about a spoon ful of the specimen, especially
that which contains mucus, pus, or blood into a clean
dry, leak proof container
• Report the appearance of the specimen
• If it is not possible to collect specimen (feaces),
perform arectal swab by inserting a cotton wool
swab into the rectum for about 10 seconds
• Label the specimen and send it with arequest form
to reach the laboratory with in 2 hrs
 In a health center for transport to a microbiology Laboratory
• Collect the specimen as described in previous text
• Transfer apportion of the specimen to a cotton wool swab
• Insert the swab in a container of sterile cary Blair
transport medium salmonella, shigella, Vibrio, and
yersinia spp. survive well in cary-Blain medium for up to
48 hrs and campylobacter for up to 6 hrs
• If cholera is suspected: Transfer about 1 ml of specimen
into 10 ml of sterile alkaline peptone water, label and
send it to reach amicrobiology laboratory with in 8 hrs
• Write the appearance of the sample on the request form
• Label and send the specimen to microbiology laboratory
with in 6 hrs
2. laboratory Examination of feaces
* on day- 1
I. Describe the appearance of the specimen
– Normal faeces: appear brown and formed or semiformed
infant faeces are yellow- green and semiformed
II. Examine the specimen microscopically
– direct wet mount preparation for ova or parasite
– Methylene blue preparation to detect fecal leucocytes
– Place a drop of methylene blue stain on a slide
– Mix a small amount of specimen with the stain
– And cover with a cover glass
– Examine the preparation for faecal leucocytes using 40x obj
– Fecal pus cells are associated with bacteria that cause
inflammation of the larg intestine (shigella, salmonella &
campycobacter)
– Those mono nuclear cells are found mainly in typhoid fever
and same parasitic infection
• Basic fuchsin smear to detect campylobacter
* Make a thin smear of the specimen on a slide
• Dry and gently heat fix it
• Cover it with 10g/l basic fuchsin for 10-20 seconds
• Examine the smear for campycobacters using the 100x
oil obj
• Motility and slide immobilization test if cholera is suspected
– Examine an alakaline peptone water culture for vibrios with a
rapid and darting motility
– The preparation is best examined using dark- field
microscopy but transmitted light can also be used
– If you see a characteristic motility, run a drop of [Link]
O-group 1 polyvalent antiserum underthe cover glass
– Re-examine for the immobilized bacteria with in 5 minutes
III. culture the specimen
If the specimen is formed or semiformed, make athick suspension
of it in about 1 ml of sterile peptone water
• Xylose lysine deoxycholate (XLD) agar and selenite broth
• Inoculate a loop ful of the emulsified faeces or fluid
specimen on XLD agar
• And several loop fuls into selemite F enrichment broth
• Incubate the XLD agar plate aerobically at 35-370C over
night
• Incubate the selenite broth at 35-370C over night
• Campylobacter medium if the patient is under two years
or campylobacter enteritis is suspected
• Inoculate a large loop full of specimen
• Place the plate in ajar or tin with a lighted candle
and incubate preferably at 420C overnight or if it is
not possible at 370C for up to 48hrs
• Note- if a selective medium is not available use a
filtration method to isolate campylobacter
• Alkaline peptone water and TCBS if cholera or
[Link] food poisoning is suspected
• Inoculate several loop fulls of specimen in alkaline (PH=
86) peptone water
• And incubate at 35-370C for 5-8 hrs
• Sub culture several loopfuls of the peptone water culture
on TCBS agar
• Incubate anaerobically at 35-370C overnight
• Note:- [Link] will also grow on TCBS at room temperature
• The medium is as selective when incubated at room
temperature as at (35-370C)
• MacConkey agar or SS agar if [Link] is
suspected
• Inoculate a loopful of specimen on MacConkey
agar or on SS agar to which has been added an
additional amount of sodium deoxycholate
(0.2g/10ml of medium)
• Incubate aerobically at room temperature (20-
280C ) for up to 48 hrs
• Examine for growth after over night incubation
• Investigation of enteritis caused by pathogenic [Link]
• Toxin tests have been developed to detect
enterotoxigenic [Link] (ETEC)
• If ETEC is suspected isolate should be subcultured on
slopes of nutrient agar and sent to microbiology lab
• If EPEC also suspected it should be sent to micro.
Laboratory
• EIEC investigation also require referral micro. Laboratory
IV. Examination and report the cultures
• XLD agar and selenite broth cultures
• Look for the appearance of shigella and salmonella
colonies
• If no colonies resembling salmonella spp. are seen
subculture the selenite broth culture on a plate of XLD
agar, and incubate at 35-370C over night
• Identification of suspect salmonella and shigella isolates
• Using a sterile straight wire, touch the surface of an
individual smooth colony, and stab into atube of MIU
medium
• Incubate at 35-370C for up to 4 hrs, examine at half
hourly intervals for a pink colour through out the medium
• A positive urease test indicates that the organism is
probably proteus no further tests are required
• If the urease test is negative at 4 hours, perform a rapid APIZ
salmonella shigella screening test as follows:-
• Place an indole strip in the neck of the MIU inoculated
tube
• Then re incubate at 35-370C overnight
• Inoculate KIA with colony from XLD plate, stab bing first
the butt and then streaking the scop.
• Close the tube with a loose- fitting cap, and incubate at
35-370C over night
• After overnight incubation, examine the MIU and KIA
cultures for reactions
• After overnight incubation, examine the MIU and KIA
cultures for reactions suggestive of salmonella or
shigella
• All strains of salmonella and shigella are urease
negative (no pink colour through out the medium)
• Test serologically any isolate giving reactions
suggestive of salmonella or shigella spp. using a
lide technique
Slide agglutination technique:
 Emulsify a small amount of growth from the KIA culture in a
loop full of physiological saline on a slide
 Mix by tilting the slide back wards and forwards for about
30 second
 Examine for agglutination aginst a dark back ground
Note:- if there is agglutination the strain is unsuitable for
serological testing
 If there is not agglutination, add one loop ful of test
antiserum and mix
 Examine for agglutination
 A+ve test will show strong clear agglutination with in 1
minute
salmonella or shigella
Note:- same times if is necessary to heat a saline suspension
of the organism in water bath for 20 minute to do O Ag testing
Rapid APIZ salmonella shigella screening test
 Add 1 drop (not less than 50µl)of sterile distilled water to
couple A & B
 Using the applicator stick provided, carefully remove a
smooth colony from the XLD agar plate and inoculate
couple A and then couple B
 Replace the tray lid and incubate at 35-370C for up to 2
hrs
 Examine cupule A for a change in color
 No colour change …………………………………..
Possibly salmonella or shigella
 Yellow, brown, green………………………………. Not
  If a red colour develops ( and the oxidase test is-ve),
suspect salmonella and test the colonies serologically
 If no red colur develops, perform amotility test using the
peptone water culture in cupule B
 If the organism is motile- suspect salmonella and if non
motile, suspect shigella
 Campylobacter medium culture
◦ [Link] and [Link] produce non haemolytic usually moist
spreading colonies on campycobacter selective medium if
growth is present, identify the colonies as follow:-
 Perform oxidase and catalase test (campycobater is +ve for
both)
 Examine abasic fuchsin smear
 Examine a physiological saline preparation for motility
TCBS agar culture
 [Link] is sucrose fermenting and there for
produces yellow 2-3mm in diameter shiny colonies on
TCBS agar with yellow colour in the medium
 And it should be differenciated from other vibrio spp.
Identification of a suspect [Link] isolate
 Examine a gram stain of the culture
 Subculture the organism on a slope of nutrient agar
(use aheavy inoculum), and incubate for 4-6 hrs
 Perform an oxidase test on the nutrient agar culture
 If positive for oxidase test, test the colony with
[Link] O group 1 polyvalent antiserum using a
slide technique
Identification of a suspect [Link]
◦ Perform an oxidase test
◦ Examine Gram stain smear of the colonies
◦ Sub culture the organism into:-
 sodium choloride peptone water
 Peptone water containing 8% w/v Nacl
 Peptone water containing 10% w/v Nacl
◦ Incubate the subcultures at 35-370C for 6-8 hrs.
10. COLLECTION, TRANSPORTATION, AND EXAMINATION OF
URINE
1. Collection and Transport of urine
• Midstream urine (MSU) for Microbiological examination is
collected as follows:-
 In a hospital with a microbiology laboratory
• Give the patient a sterile, dry, wide- necked, leak proof
container , and explain to collect a clean catch specimen
• Female patients should be instructed to cleanse the area
around the urethra openings with clean water, dry the
area and collect the urine with the labia held apart
• About 20 ml of urine should be collected
• Label and deliver the specimen to microbiology Lab. As
soon as possible
• If delay indelivery is expected refrigerate the sample at
40C, if the delay is more than a hour is anticipated boric
acid should be added
• Note:- Urine for culture should be preserved with
bactericidal chemical
• Label and send it with the request paper to reach
microbiology laboratory with in 48 hrs
 Collection of urine if renal tuberculosis is suspected
• Collect the first urine passed (entire specimen) on three
successive morning
• The collection container should be clean, dry , leak- proof
and sufficiently large to contain all the first passed
morning urine
• The specimens should be stored at 4 0C until all three
urines have been collected.
2. Laboratory examination of urine
* Day- 1
• Describe the appearance of the specimen
• Report:- color,whether it is clear or cloudy
– Normal freshly passed urine is clear and pale yellow to
yellow depending on concentration
– When left to stand, a cloudiness may develop
Examine the specimen microscopically
 Wet preparation of fresh uncentrifuged urine
 In the investigation of urinary infection, it isef greater value to
look for bacteria in fresh un centrifuged urine than estimating the
number of pus cells in sentrifuged urine
 Place three loop full of well- mixed fresh urine on a slide, and
cover with a cover glass
 Examine the preparation using 10x and 40x objectives with the
condenser iris closed sufficiently to give good contrast.
The following may be found in urine:-
 Bacteria
 Pus cells
 Red cells
 Yeast cells
 Epithelial cells
 Parasites,etc
The finding of bacteria in a freshly passed uncentrifuged urine
indicates urinary infection
Gram smear
 Centrifuge about 10 ml of urine
 Discard the supernatant fluid, and make a smear of the sediment
 Fix and stain by Gram technique
 Examine it under oil immersion objective
 Look especially for bacteria associated with urinary infection,
especially gram –ve Rods
 Vaginal contamination is indicated by a mixed bacterial flora,
including G+ve rods.
 Neisseria gonorrheal in urine
 In male patients with acute urethritis, it Is often possible to make
a presumptive diagnosis of gonorrhoea by finding G-ve intra
cellular diplococci in pus cells
 Dark – field preparation if lep tospirosis is suspected
 The examination of leptospires can be done using dark field
Microscopy
 Ziehl-Neelson smear if renal tuberculosis is suspected
• Make a smear of the deposit from three centrifuged early
morning urine sediments
• Allow to air dry and fix it with 70%v/v alcohol
• Make AFS stain
• Examine as described in the previous text for AFB.
• Culture the specimen
• culture is required if the urine contains bacteria, cells, casts,
protein Nitrite, or has a markedly alkaline or acid reaction
 Estimating bacterial numbers
• The approximate number of bacteria per ml of urine, can be
estimated by using a calibrated loop or a measured piece of
filter peper
• Both methods are based on accepting that a single colony
represents one organism
• For example, if an inoculum of 1/500 ml produces 20 colonies,
the number of organisms represented in 1/500 ml of urine is 20
or 10,000 in ml (500x 20)
•
 Blood agar and MacConkey agar
 Mix the urine well by inverting the container several times
 Using a sterile calibrated wire loop inoculate a loop full of urine
on blood agar and MacConkey agar
 The loop must be held vertical and only the loop must be dipped
in the urine
 Incubate the inoculated plates aerobically at 35-370C over night.
 Selenite broth to detect [Link] carriers
 Centrifuge 7-10 ml of urine in a sterile conical tube at high speed
for 5-10 minutes
 Discard the supernatant fluid and resuspend the sediment
 Transfer the entire sediment to a container of selenite
enrichment broth
 Incubate the broth at 35-370C overnight
 The following morning, subculture on XLD agar
 And incubate the plate acrobically at 35-370C for 24 hrs

 Lowen stein Jensen slope if renal tuberculosis is suspected
• Collect 3 early morning urines
• Allow each urine to sediment overnight (40C)
• Discard the supernatant from each tube and combine the
sediment
• Centrifuge the sediment at high speed for 20 minute
• Discard the supernatant fluid and resuspend the sediment
• After making AFS preparation , mix the sediment with
equal volume of NaoH (40g/l) soln and mix
• Leave the mixture for 10 minutes to decontaminate the
urine
• Inoculate 200µl (0.2 ml) of the well- mixed
decontaminated urine on the scop of LJ media
• Incubate the inoculum as described previously for AFB.
3. Examine and report the culture
• On blood agar and MacConky agar cultures look
especially
for colonies of : -
• [Link]
• Proteus spp.
• [Link]
• Klebsiella strains
• [Link]
• Enterococci
• Note:- contaminating organisms usually produce a few
colonies of mixed growth
• Estimation of bacterial numbers count the approximate number
of colonies. Estimate the number of bacteria per ml of urine
• Report the bacterial count as:- less than 10000/ ml 
contamination
• 10,000-100,000/ml  infection
or contamination
• More than 100,000/ml infection
• XLD agar culture
• Look for colonies of [Link]
• LJ culture
• Look for colonies of [Link]
11. COLLECTION, TRANSPORT, AND EXAMINAITON OF
CEREBROSPINAL FLUID (CSF)
1. Collection of CSF
• it should be collected by an experienced medical officer
or Health worker
• It must be collected as aseptically as possible to prevent
organisms being introduced into the central nervous
system
• The fluid usually collected from the arachoid space
• A sterile wide- bore needle is inserted between the fourth
and fifth lumbar vertebral
• The C.S.F is allowed to drip into a dry sterile container
 In a hospital with a microbiology laboratory
• Laboratory staff should be advised before a lumbar
puncture is performed so that they can be
prepared to receive and examine the specimen
immediately
• Note: A delay in examining CSF reduces the chances of
isolating a pathogen. It will also lead to a falsely low
glucose value due to glycolysis
• Take two sterile, dry, screw- cap containers and
label one No.1 (first sample collected, to be used
for culture), No.2 (second sample collected, to be
used for other investigations
• Collect about 5 ml of C.S.F in container No.1 and
about 2-3ml in container No.2)
• Deliver immediately the samples with a request
form to the laboratory
2. Laboratory examination of cerebrospinal fluid
* Day- 1
I. Describe the appearance of the specimen
 Whether the C.S.F is clear, slightly cloudy, cloudy, or
definitely purulent
Note: If the C.S.F is purulent or markedly cloudy, make
immediately a smear for Gram staining, and report it as soon as
possible
 Whether if contains blood and if so whether sample No.2
contains as much blood as sample No.1
 Sample No.1 contain more blood to traumatic lumber
puncture
 Sample No.1 and 2 due to haemorrhage in the CNS
 The C.S.F may appear xanthochromic (yellow- Red) due to
sub arachnoid haemorrhage or Jaundice or spinal
constriction.
When it contains clots
 clotted C.S.F indicates an increase in fibrinogen
 It occurs when there is spinal constriction
 It can also occur in pyogenic meningitis
 In tuberculosis meningitis a skin may form on the
surface of the fluid
Normal C.S.F:- appears clear, bright and colurless
II. Perform a cell count
– Using a dry pateur pipette held vertically, transfer 3 drops of
well- mixed C.S.F (sample No.2) to small dry tube
– Using another Pasteur pipette of the same bore size, add 3
drops of toludine blue diluting fluid, and mix well
– This gives a 1 in 2 dilution of the C.S.F
– Count the cells using an improved neubauer chamber under
10 x objective with condenser, iris closed sufficiently
– Count the cells in 4 of the large squares
– Then multiply the number of cells counted on the 4
squares by 2 if using 1 in 2 dilution; then multiply the
figure obtained by 10/4
– Multiply by 106 to give the number of cells per litre of
C.S.F
– Normal C.S.F contains up to 5x106 cells/ lit
– In meningitis and other diseases, the C.S.F contains
more than 5x106 cells/ litre
III. Test the specimen biochemically
◦ centrifuge No.2 sample at medium to high speed for about 5
minutes
◦ Transfer the supernatant fluid to another tube
◦ Use the clear supernatant fluid for biochemical tests , and the
sediment for the microscopic examination
IV culture the specimen (sample No.1)
◦ Culture of the C.S.F is necessary if the fluid contains cells
and ,or, the protein concentration is abnormal
Important:- C.S.F should be cultured as soon as
possible after collection
 If a delay is un avoidable, the fluid should be kept at
35-370C (never refrigerated)
 If the C.S.F appears only slightly cloudy, centrifuge
it in a sterilie tube for 15-20 minutes and use the
sediment for inoculating the plates
 Chocolate (heated blood)agar
 Inoculate the specimen on chocolate agar
 And incubate it in a Co2 enriched atmosphere at 35-
370C for up to 48 hrs, checking the growth after
over night incubation
 MacConkey agar and blood agar if the patient is a New
born.
 Inoculate the specimen on MacConkey agar and blood
agar
 Incubate both plates at 35-370C over night
 LJ medium if tuberculosis meningitis is suspected
 Inoculate the C.S.F on a slop of LJ medium
 Incubate it
Note:- It is usual to wiat until the following morning to check
whether the chocolate agar culture is sterile before
inoculation of the LJ medium
- If the culture is not sterile, decontaminate the sediment
by adding of 4% w/v NaoH . then inoculate it.
 Sabouraud agar if cryptococcal meningitis is suspected
 if capsulated yeast cells are seen in the microscopical
preparation, inoculate a plate of sabouraud agar
 Incubate at 35-370C for up to 72 hrs
 Checking the growth after overnight incubation
V. Examine the specimen Microscopically
• The microscopic examination of C.S.F is required if the specimen
appears abnormal, contains cells and , or, the total protein is raised
with a positive pandy’s test
 Gram smear
• Make an evenly spread smear of a drop of purulent C.S.F ., or
the sediment from a centrifuged sample (No.2) on a slide
• And allow to air dry in a safe place
• Fix with methanol, and stain by the gram technique
• Examine the smear for pus cells and bacteria using 40x and
100 x objectives
• Look for:-
– G-ve intracellular diplococci ……..> N .meningitides
– G+ve diplococci or short streptococci …….> [Link]
– G- ve rod, especially if filamentous ………..> H. influenze
– G+ve cocci ingroup or singley……………….> S. aureus
– G+ve strep. Cocci………………………………..>
[Link]
– G+ve yeas cells …………………………………..>
[Link]
 AFB stain
 If tuberculosis meningitis is suspected, transfer several
drops of C.S.F sediment to a slide, allowing one drop to dey
before adding the next
- Note:- AFB are difficult to detect in C.S.F the chances of
finding the bacteria are increased if C.S.F is centrifuged for
20-30 minutes and several drops are used
 Allow the smear to air dry in a safe place
 Fix with methano and stain by ziehl Neelsen Method- I
 Then examine under 40x obje & 100x objective
 India ink preparation if cryptococcal meningitis is
suspected
 Transfer a small drop of C.s.F sediment to the slide,
 Add a small drop of India ink or 20% nigrosin
 Mix, and cover with a cover glass
 Examine the preparation under 40x objective
 Look for oval or round stained cell cells measuring 5-
20µdm
 If cells resembling [Link] are seen culture
the C.S.F on sabourand agar
 Notify immediately the medial officer attending the
patient
 Wet preparation to detect
• Naegleria amoebae
• Trypanosomes
 Do Giemsa smear to detect
VI Examine and report the cultures
* on Day- 2 and on wards
 Chocolate agar culture
– Look especially for colonies that could be:-
• [Link]
• S. pneumoniae
• H. influenzae

MacConkey agar and blood agar cultures.
– Look especially for colonies that could be:-
• [Link] or other coliform
• [Link]
• [Link]
• [Link]
 LJ-culture
– Look for [Link]
 Sabouraud agar culture
– Look for C. neoformans
12. COLLECTION, TRANSPORT, AND EXAMINATION OF
BLOOD AND BONE MARROW
1. Collection and culture of blood and bone marrow
 Blood and bone marrow require culturing immediately after
collection before clotting occurs
2. Choice of culture media
◦ The following media are suitable for the routine culture of
blood and bone marrow
 Tryptone soya (tryptic soy) diphasic medium
 Thioglycollate broth medium
3. Examination of blood and bone marrow
* Day- 1
I. Collect and culture the specimen
◦ Blood :-
 Blood should be collected before antimicrobial treatment
has been started and at the time the patient’s temperature
is beginning to rise
 To increase the chances of isolating a pathogen, it usually
recommended that at least two specimens (collected at
different times should be cultured )
 Blood for culture should be collected as aseptically as
possible the following technique is recommended:
 – Using a pressure cuff, locate a suitable vein in arm
– Cleanse thoroughly the skin over the vein using tincture of
iodine followed by ethanol- ether
– Remove the protective cover from the tops of the culture
bottles and cleanse the top of each bottle using an ethanol-
ether swab
• Important:- Do not use a bottle of culture medium if it shows
signs of contamination, i.e forbid broth
- Do not use a bottle of thioglycollate broth if it appears
oxidized
• Using a sterile syringe and size 21 gauge needle, with draw
10-12ml of blood
• With care, remove the needle from the syringe, and replace
with another sterile needle of similar size
• Insert the needle through the rubber liner of the both the cap
and dispense 5 ml of blood in to each culture bottle dispense
the remaining blood in to a tube or bottle containing EDTA for
TLC and parasite examination
• Using an ethanol- ether swab, wipe the top of each culture
bottle and replace the tape or protective caps. Gently mix the
blood with the broth
Important: the blood must not be allowed to clot in the
culture media because any bacteria will become
trapped in the clot
Using alead pencil- label each bottle with the name and
number of the patient, and the date and time of collection
As soon as possible, incubate the inoculated media
◦ thioglycollate broth
 Incubate at 35-370C for up to 2 weeks
 Examine and cubculture
 Tryptone soya diphasic medium
 Incubate at 35-370C for up to 4 weeks
 Examine for growth
N.B if brucellosis is suspected, loosen the cap of the
diphasic culture bottle or insert a sterile needle through
the rubber liner, and incubate in a CO2 atmosphere
Culture of blood from new born infants
◦ To reduce the risk of contamination, blood from
neonates should be collected from a peripheral vein
not from the umbilical vein
Bone marrow (BM)
◦ The aspiration of bone marrow is usually performed by
a medical officer
◦ Using a strict aseptic technique, inoculate the BM in to
tryptone soya diphasic medium
Note:- If only a small amount of marrow is obtained, add
aseptically 3-5 ml of steile nutrient both to the inoculated broth
– Incubate the inoculated diphasic medium as described for
blood cultures
– Make a smear of the BM for Giemsa staining
II. Examine the specimen Microscopically
• Giemsa smear
– Blood
• Centrifuge a sample of the EDTA anticoagulated blood,
and make a smear of the buffy coat layer
• When dry, fix with methanol for 2 minutes, and stain by
the Giemsa method
• Examine the smear for malaria parasites and if indicated,
look also for barreliae, microfilariae, and trypanosomes
 – BM
• Examine the BM for, malaria, [Link], Microfilariae,
trypansomes

• Dark field preparation

• If leptospirosis is suspected, centrifuge a sample of fresh
EDTA blood and examine a drop of the plasma for motile
leptospires using dark field microscop
• Thloglycollate both culture
• Examine daily for up to 14 days
• Look for visible signs of bacterial growth such as turbidity
above the red cell layer, colonies growing on top of the
red cells (cotton balls) haemolysis, gas bubbles, and clots
• A sterile culture usually remains clear. A slight turbidity
may develop after several days incubation
• If there are signs of bacterial growth, subculture the broth
and examine a Gram stained smear for bacteria
• Important: if the patient is seriously ill, subculture the
broth (even in the absence of visible bacterial growth)
after overnight incubation, after 48hrs, and twice weekly
for up to 2 weeks.
sub culturing a blood culture broth
 A strict aseptic technique must be used to avoid
contaminating the culture
 Using an etanol- ether swab, cleans the top of the bottle
 Using a sterile needle and small syringe, insert the needle
through the rubber liner in the cap and with draw about 1 ml
of the broth culture
 Inoculate the broth on :- Blood agar ,Chocolate agar &
MacConkey agar
 Swab the top of the culture bottle, and reincubate
Tryptone soya diphasic media:
 Examine daily for the first of days and twice a week for up
to 4 weeks
 Look for colonies on the agar slope (preferably using a
hand lense ) and signs of bacteria growth in the broth
If growth is present:-
 Sub culture on blood agar, chocolate agar and
macConkey agar
 Examine a Gram stained smear of the colonies
 Depending on the colonies examine for- coagulase,
catalase oxidase, urease and motility
 If large G+ve rods resembling [Link] are seen,
subculture also on lactose agg yolk milk agar and incubate
the plate anaerobically
 If motile, urease and oxidase -ve, G-ve rodes are isolated,
subculture the colonies on KIA to screen for salmonella
 If catalase positive, G-ve coccobacilli are isolated from a
diphasic culture that has been incubated in Co2
atmosphere, suspect Brucella spp.
Important:- Always report immediately a positive blood culture,
and send a preliminary report of Gram smear and other
usuefull test results
 Reference
1. Monica Cheesbrough, District LaboratoryPractice in Tropical Countries
Part II. 2nd ed. 2006.