Selection, collection, and transport of specimen to the laboratory is an essential part of quality assurance of the microbiologist .

1. Prepared by:
Jehad Jamil Obaid
Microbiology department- central lab.
y
Advanced Specimen Collection
and Culture Work-up

2. Introduction

3. Introduction
 Good identification of the isolates and the knowledge of its behavior
as commensals or pathogens play a vital role in the admission
(starting and choosing) of treatment for the cure of the patient and
controlling for nosocomial infection.
 selection, collection, and transport of specimen to the laboratory is
an essential part of quality assurance of the microbiologist in other
hand.
 Laboratory professionals must promote, advice, and collaborate in
taking measures to ensure proper selection, collection, and
transport of specimen.

4. Laboratory Safety Rules
These rules are for the safety of the instructors and medical staff
1. Wear a lab coat.
2. No eating or drinking during lab.
3. Keep long or fluffy hair tied up and out of the way.
4. Always wear shoes in lab.
5. Thoroughly wash your hands with soap and water before and
after work.
6. Clean the lab bench with disinfectant before and after work.
7. Keep the lab bench free of unnecessary materials.
8. Do not take cultures from the lab area.
9. Dispose of all contaminated materials in autoclave.

5.  Every specimen must be evaluated for its suitability
for processing.
 The general criteria for rejection of specimens are
listed below:
1. Un labeled specimens.
2. The information on the label does not match the information
on the request form.
3. The specimen was transported in an improper container or at
wrong temperature.
4. The quantity of the specimen is insufficient to Cary out all the
required examination.
5. Leaking specimen.

6. Type of Culture
 Blood culture
 Urine culture
 Ear culture
 Conjunctival culture
 Pus and burn culture
 CSF culture
 Throat culture
 Sputum culture
 Vaginal discharge
culture
 Genital culture
 Stool culture

7. Blood Culture

8. Blood Stream Infection
Common pathogens
Bacteroides fragilis and other
anaerobic bacteria
Streptococcus spp
Coagulase negative staphylococciStaphylococcus aureus
Enteric gram negative bacilliListeria monocytogenes
Neisseria meningitidesAcinetobacter spp.
Non fermenter gram negative bacilliHaemophilus influenza
Pseudomonas aeruginosaSalmonella typhi

9. Aim of the test
 An etiological diagnosis of bacteremia by aerobic and anaerobic
cultivation of the blood, with identification and susceptibility test of
the isolated organism(s).
Types of specimen
 Whole blood.
Criteria of specimen rejection
 Blood collected in tubes or bottles other than aerobic and
anaerobic blood culture bottles.
 If the information on the label does not match that of the request
form.
 Specimens for anaerobic blood culture received in aerobic bottles or
vice versa.
Blood Culture

10. All percussion should be taken to minimize the percentage of
contaminated blood culture, to reduce the chance of
contaminating organisms from the skin the vein puncture site
should ideally be prepared as follows;
1. Wash with soap, rinse with sterile water or saline.
2. Apply 1-2 %During blood tincture of iodine or povidone –iodine
and allow drying for 1-2 minutes.
3. Remove the iodine with 70 % alcohol wash, if the site again be
pulpated after the iodine – alcohol preparation the finger must be
disinfected or sterile gloves worn.
4. A tourniquet is applied to the upper arm above the vein puncture
site to distend the anticubital veins.
Specimen collection

11.  Before starting antibiotics therapy if time permits, its generally
recommended that the first two sets of blood cultures be taken
one hour apart and the third set after 3-6 hours.
Volume of Blood Culture Collected According to Age Of Patients
Age of patient No. of blood bottle
Children below 2 years 1 mL of venous blood in 2 bottles
Children 2-5 years 2 mL of venous blood in 4 bottles
Children 6-10 years 3 mL of venous blood in 4 bottles
Children 11-15 years 5 mL of venous blood in 4 bottles
Children above 15 years and adults 5 mL venous blood in 3 sets of bottles (6 bottles).
Collection Time

12. Remove Flip Caps from the tops of the selected culture
bottles. Disinfect the septa of the bottles with alcohol or
iodine preparation and allow to dry.
Perform venipuncture with syringe and collect the
desired amount of blood. If the vein is missed a new needle
should be used.
Transfer the recommended amount of blood into the
culture bottles using aseptic technique if desired. First fill
the aerobic bottle. Do not overfill the bottles! Any
remaining blood may be used for additional tests.
Label the bottles according to the routine procedure.
When using a sticker do not cover the tear-off section of
the barcode label .
Note: 1:5 to 1:10 blood/broth ratio is the appropriate ratio to achieved, this dilution
minimizes the effects of microbial inhibitors present in blood and dilutes any antimicrobial
agents.
Collection of Blood for Culturing

13.  Although coagulase-negative Staphylococcus is the most
commonly isolated organism from blood cultures, only a few
(6.3%) of the isolates represent "true" clinically significant
bacteremia.
 The physician is responsible for determining whether an
organism is a contaminant or a pathogen .

14. The number of blood culture:
1. 80% of these are detected with the first culture and 99% within
the three cultures.
2. More than three cultures are therefore not necessary unless the
patient has received antibiotics, in this case a new series of blood
cultures maybe indicated after the antibiotics treatment has been
stopped.
Specimen processing :
1. The bottle incubated for 24 hour before plating to enhance the
growth of bacteria.
2. aerobic bottle plate on blood agar, MacConky, and chocolate in
CO2 incubator for 24 hour.
3. anaerobic incubate anaerobically on blood agar for 48 hour .
4. the negative bottle should be reincubated and tested after one
week before discarded as negative culture.

15. Specimen processing
The anticoagulant in blood culture medium must not
harm the bacteria and must prevent clotting of the blood,
which entrap bacteria and prevent their detection .
The most commonly used preparation in blood media is
0.025% to 0.05% Sodium polyanethol sulphonate (SPS).
If slow growing organisms are suspected as Brucella spp.
its should be clearly indicated on the requisition form and
the culture bottles should be further incubated for 2-4
weeks before being reported out as negative.

16. A negative culture result does not necessarily rule out bacteremi a;
false-negative results occur when pathogens fail to grow.
A positive culture result does not necessarily indicate bacteremia;
false-positive results occur when contaminants grow.
Post specimen processing
Result reporting
Any isolated organism will be reported. Antibiotic sensitivity will also be
included with the report.
Turn around time
 Initial blood culture results will be reported as soon as it shows growth.
 Final results with sensitivity will be issued after 24- 48 hours of the
initial report.
 Negative results will be issued after 10 days of culture submission.

17. Urine Culture

18. Urinary Tract Infection

19. Commensal floraCommon pathogens
Diphtheroid bacilliNeisseria gonorrhoeae
Lactobacillus sppE.coli and other Enterobacteriaceae
Coagulase negative StaphylococciEnterococcus spp
Alpha Haemolytic StreptococciStaphylococcus aureus
Bacillus sppStaph saprophyticus
Anaerobic cocciAcinetobacter spp
Commensal MycobacteriumPseudomonas spp
Gardnerella vaginalis
Beta -haemolytic streptococci
Salmonella spp (early stage of infection)

20. Specimen Collection
1. Collection of midstream urine for bacterial investigation.
2. Catheter Collection
 Catheterization for the expressed purpose of obtaining urine specimens
should be avoided if possible because of the high risk of introducing
nosocomial infection .
 The first several milliliters of urine from the catheter should be
discarded to wash out any organisms that may have lodged in the
catheter tip during transit through the urethra .
 Urine sample shouldn’t be obtained from catheter bag except from
neonates or young infants when special precaution have been taken .
3. Suprapubic Aspiration
 Suprapubic aspiration are reserved almost exclusively for neonates,
small children, and occasionally for adult with clinically suspected
urinary tract infection in whom clean catch samples have failed to
establish a diagnosis.

21. Specimen processing

22. 1. Mix the urine sample to resuspend microorganism present.
2. Dip a 1 μl calibrated loop in vertical position in the urine and
remove the loop and use the collected fluid to inoculate a
nutrient agar plate that will be used for urine plate count.
3. Then take another loop to streak Blood agar and another loop to
streak MacConkey agar plates.
4. Some laboratory prefer to inoculate of duplicate plates with both
1 &10 μl calibrated loops for comparing of counts as equality
control check.
5. When fastidious organism such as Neisseria gonorrhoeae is
suspected an enriched medium such as chocolate agar required
determining the cause of infection.
Specimen Processing

23. 6. After incubation, a colony count of 10 5 CFU/ ml (colony
forming unit per milliliter) or higher is the criterion used to
determine if organisms identification and susceptibility testing
are to be performed.
7. When the colony count is between 104 and 105 CFU/ml or
when two or more species are recovered, the decision to
make identification and perform susceptibility tests must be
made on an individual case by case basis.
8. Culture of catheterized or suprapubic urine specimens are
usually analyzed in detail, even with low colony counts or with
recovery of multiple organism types.
9. Colony counts as low as 102 CFU/ml of enteric Gram negative
bacilli may be significant in female patients with the acute
uretheral syndrome, however the physician must alert the
laboratory in suspected cases, because semiquantitative urine
culture techniques are not designed to detect such low colony
count and will be signed as non significant growth.

24. Mix the urine sample to re-suspend microorganism present.
Dip a 1 μl or 10 μl calibrated loop in vertical position in the urine and
remove the loop and use the collected fluid to inoculate Nutrient, Blood
and MacConkey agars respectively.
Culturing Procedure

26. Urine
Gram PositiveGram Negative
Penicillin GAmoxycillin/ Clavulanate
AmoxycyclineAmpicillin
AmpicillinCefaclor
CefuroximeCefuroxime
TetracyclineTetracycline
DoxycyclineDoxycycline
CiprofloxacinCiprofloxacin
OfloxacinOfloxacin
Sulfamethaxazol/TrimethoprimSulfamethaxazol/Trimethoprim
GentamicinNitrofurantoin
ErythromycinNalidixic Acid
CephalexinNorfloxacin
Amoxycillin/ClavulanateAmikacin
Cloxacillin

27. Throat Culture

28. Aim of the test
A throat swab culture is a laboratory test done to isolate and
identify organisms that may cause infection in the throat
mainly group A beta-hemolytic streptococci.
Types of specimen
Two Swabs from posterior pharynx, tonsils, or other inflamed
area.
Criteria of specimen rejection
Inappropriate specimen transport device, mislabeled
specimen, Unlabeled specimen, Dried samples.
Specimen received after prolonged delay (usually more than 2
hours).
Specimen received in expired transport media.
Throat Culture

29. Collection of Specimen
Throat Swabs
1. Turn the patients face against the light, ask the patient to open his
mouth wide and phonate an “ah” gently depress the patients
tongue with a tongue blade so that the throat is well exposed and
illuminated.
2. Guide a swab over the tongue into the posterior pharynx.
3. Rub the swab firmly over the back of the throat, both tonsils and
any areas of inflammation, exudation or ulceration. Care should be
taken to avoid touching the tongue, cheeks or lips with the swab.
4. Place the swab in the transport medium and push it down to the
bottom.

30. Specimen processing
Because Streptococcus pyogenes is the primary case of
pharyngitis most laboratories routinely screen throat
cultures for this organism.

31. Commensals floraCommon pathogens
Enterobacteriaceae and
other than the common pathogens
Helicobacter pylori
Bacteroides sppSalmonella spp.
Streptococcus sppE. coli O157:H7
LactobacilliStaph aureus
Pseudomonas spp.Campylobacter spp.
Coagulase negative staphylococciVibrio cholerae
BacteroidesYersinia enterocolitica
ClostridiumClostridium difficile
PeptostreptococcusShigella spp.
Bifidobacterium
Eubacterium.
Upper Respiratory Tract Infection

32. Nasopharyngeal Swab
1. Have the patients head firmly supported.
2. Insert a nasal speculum.
3. Gently insert a wire swab through the nostrils to the
posterior nasopharynx.
4. Rotate the wire swab gently and allow it to remain in
that position for 20-30 seconds, and then withdraw
deftly.
5. Place the swab in the transport medium

34. Specimen Processing
 Culture
1. Because Streptococcus pyogenes is the primary case of
pharyngitis most laboratories routinely screen throat
cultures for this organism.
2. Classically throat swabs plated on 5% sheep blood agar
plates, streak the swab across blood agar plate to make a
line that divide the plate into two halves and using a sterile
loop streak by crossing the line to produce isolates
colonies .
3. make few stabs in the agar plates .
4. Inoculate another Chocolate and MacConkey agar plates
also are recommended if organisms other than S. pyogen
is suspected.

35. Infection of the Lower Respiratory Tract

36. The common pathogens
Haemophilus influenzaeStreptococcus pneumoniae
Klebsiela pneumonia and
other Enterobacteriaceae
Staphylococcus aureus
Mycobacterium spp.Moraxella catarrhalis
Bordetella sppFusobacterium spp
Legionella spp.Chlamydia pneumoniae

37. Specimen Collection
Expectorated sputum
 All expectorated sputum is contaminated to some degree
with secretion of the oropharngeal cavity.
 Early morning sputum samples should be obtained because
they contain pooled overnight secretions in which pathogenic
bacteria are more likely to be concentrated.
 Instruct the patient to brush his teeth and gargle with water
immediately before obtaining the sputum specimen to
reduces the number of contaminating oropharyngeal
bacteria.
 To prevent contaminated of the out side of the container the
patient should be instructed to press the rim of the container
and the lower lip to catch the entire expectorated cough
sample.

38. Translarengeal (Transtracheal) Aspiration
 Translaryngeal Aspiration may be indicated when
 The patient is depilated and cannot spontaneously
expectorate a sputum sample.
 Routine sputum sample have failed to recover a causative
organism in the Face of clinical bacterial pneumonia.
 An anaerobic pulmonary infection is suspected.
Branchoscopy
 in patient with lung abscess or other suspected deep
pulmonary infection.
 uses a telescoping double catheter plugged with
polyethylene brush.
 the technique is recommended for the optimal recovery of
aerobic and obligate anaerobic bacteria from deep-seated
pulmonary lesions

39. Specimen Processing
. Direct Visual Specimen
1. Gram stain important to evaluate the realty of the
sputum specimen .
2. an acceptable specimen yield less than 10 sequamous
epithelial cells per low power field .
3. the number of white blood cells may not relevant
because many patients are severely neutropenic and
specimens from these patients will not show WBC on
gram stain examination.
4. the presence of 25 or more polymorphonuclear
leukocytes per field, togather with few sequamous
epithelial cells implies an excellent specimen.

40.  In addition to gram stain respiratory specimens may be
stained for acid fast bacilli with either classic Ziehl-Neelson or
the Kinyoun carbolfuchsin stain. Auramine or Auramine-
rhodamine is also used to detection acid-fast organisms.
Routine Media
 blood agar, chocolate agar and MacConkey agar plate.
 Transtracheal and percutaneous lung aspiration material
maybe inoculated to enrich Thioglycolate.
 bacterial agents that cause lower respiratory tract
infections are not detected by routine bacteriologic
culture as Mycobacterium, Chlamydia, and Bordettella
required especial procedure for detection.

41. Specimen processing for Sputum

42. Acid Fast Stain
General Format:
 Primary Stain (Carbol Fuchsin)
 Decolorizer (acid alcohol)
 Counterstain (Methylene Blue)

43. Principle of Acid Fast Stain

44. Examination of Acid Fast Smear
 Under oil immersion, examine at least 100, but
up to 300, useful, representative fields
(minimum time 5 mins.)

45. Number of bacilli seen on a smear Results
reported
No AFB Per 100 oil immersion field 0
1-9 AFB Per 100 oil immersion field Scanty
10-99 AFB Per 100 oil immersion field 1+
1-10 AFB Per 1 oil immersion field (min 50 fields) 2+
< 10 AFB Per 1 oil immersion field (min 20 fields) 3+
Reporting on AFB Microscopy

46. Result Reporting of
AFS

47. Pleural, Peritoneal, Pericardial
and Synovial fluids Culture

48. Aim of the test
Isolate and identify pathogenic organisms from normally sterile body
fluids and perform sensitivity test.
Infection Of Sterile Body Fluid: all body fluid are sterile
Types of specimen
Aseptically aspirated body fluid (e.g., , synovial, peritoneal fluid).
Criteria of specimen rejection
Inappropriate specimen transport device; mislabeled specimen;
unlabeled specimen; specimen received after prolonged delay (usually
more than two hour); specimen received in expired transport media.
Pleural, peritoneal, pericardial and
synovial fluids culture

49. Specimen processing
 If fluid has been concentrated by centrifugation, the resulting sediment
should be incubated to inoculated to an enrichment broth, blood,
chocolate and MacConkey agars.
 All fluids should be processed for direct microscopic examination, in
general if one organism is seen per oil immersion field at least 105
organisms per milliliter of specimen are present.
 Specimens for fungi should be examined by direct wet preparation or
by preparing 10% KOH for visualization of fungi element from a wet
preparation.
 Acid Fast stain for Mycobacterium spp.

50.  Result reporting:
 Report Gram stain, KOH, and AFS finding as an initial
report.
 Report the isolated pathogen and its sensitivity pattern as
a final report.
 Turn around time:
 Gram stain and wet mount results should be available 1
hour after specimen receipt.
 Isolation of a possible pathogen can be expected after 2-4
days.
 Negative culture will be reported out 1-2 days after the
receipt of the specimen.
Post specimen processing

51. Cerebrospinal Fluid Culture

52. Infection of Cerebrospinal Fluid
Common bacterial pathogen
Salmonella (rare)Haemophilus influenzae
Brucella (rare)Neisseria meningitis
Treponema pallidum (rare)Streptococcus pneumoniae
Listeria monocytogenesGroup A & B streptococci

53. Who will collect the specimen
 Physician
Quantity of specimen
 Mini. 5-10 ml of CSF is recommended for culture.
Time relapse before processing the sample
 CSF is an emergency specimen and should be processed
immediately.
Storage
 Maintain specimen at room temperature. Do not
refrigerate.
Pre specimen processing

54. Specimen Processing
 CSF is an emergency specimen and should be processed
immediately.
 Centrifuge clear specimen and inoculate two blood agar plates
(one for aerobic and one for anaerobic) one chocolate, one
MacConkey plate, one Thioglycolate tube.
 Examining stained smears of CSF sediment and performing
direct antigen detection test maybe helpful both in
establishing a presumptive diagnosis and in providing
guidelines for the selection of culture media.
 Microorganisms can often be detected in gram stained or
methylene blue.

55. Specimen processing

56. Some rules concerning CSF Examination
1. The specimen must be collected under sterile conditions, sealed
immediately to prevent leakage or contamination, and sent to the
laboratory without delay.
2. Blood sample should be collected 30 min. before lumber puncture
for glucose, protein and immunoglobulin determination.
3. Specimens are usually collected in three sterile tubes, labeled 1, 2,
and 3 in the order in which they are drawn, tube 1 for chemistry
and serology, while tube 2 is used for cell count and differential,
and tube 3 for microbiology (2-4 ml in each tube).
4. CSF specimens for additional chemical and serological tests should
be frozen, hematology tubes are refrigerated, and microbiology
tubes remain at room temperature.

58. Ear Culture

59. Aim of the test
Etiological diagnosis of otitis externa or otitis media by
aerobic and anaerobic culture with identification and
susceptibility test of the isolated organism(s).
Types of specimen
Two swabs from the external or aspiration from middle
ear(s).
Criteria of specimen rejection
Inappropriate specimen transport device; mislabeled
specimen; unlabeled specimen; specimen received after
prolonged delay (usually more than two hour); specimen
received in expired transport media.
Ear Culture

60. Infection of Ear
Commensalisms flora
are present in the external ear canalcommon pathogens
Staphylococcus epidermidisStaphylococcus aureus
Lactobacillus spp.Streptococcus pyogenes
Propionibacterium spp.Pseudomonas aeruginosa
Staphylococcus aureusOther Gram negative bacilli
Various EnterobacteriaceaeStreptococcus pneumoniae
Various streptococcus sppHaemophilus influenzae
Candida spp. other than albicansAnaerobic bacteria
Occasion Pseudomonas aeruginosaProteus spp.

61. Specimen processing
for Otitis external
Specimen processing
for Otitis media

62. Who will collect the specimen:
 Medical technologist, Microbiologist for swab from
external ear.
 Otolaryngologist for aspiration from middle ear.
 Who is authorized to order the test
 Physician.
Time relapse before processing the sample
 Not more than 2 hours.
Storage
 Refrigerated (2-8) 0C .
Pre specimen processing

63.  Specimen Collection
1. Collect a specimen of the discharge on a thin, sterile
cotton wool or Dacron swab.
2. Place the swab in a container with the transport
medium, breaking off the swab stick to allow the
stopper to be replaced tightly.
3. Label the specimen and send it to the laboratory.
 Specimen Processing
1. culture should be inoculated to blood agar, MacConkey,
and chocolate agar plates.
2. Direct visual examination for material aspirated from
the middle ear or mastoid is also examined directly for
fungi.

64. Pus and wound Culture

65. Aim of the test
To isolate and identify aerobic and anaerobic pathogenic
organisms from pus specimen and sensitivity test.
Types of specimen
Swabs from the infected area or aspiration from deep wounds.
Swabs in anaerobic transport media for the isolation of
anaerobes.
Criteria of specimen rejection
Inappropriate specimen transport device; mislabeled specimen;
unlabeled specimen; specimen received after prolonged delay
(usually more than 72 hours); specimen received in expired
transport media and dried samples.
Pus and wound Culture

66. Skin Pathogen and Commensals
Commensals flora
Staphylococcus epidermidisDiphtheroides
Other coagulase negative staph.
Pathogenic bacteria
group A StreptococcusStaph aureus
Bacillus anthracisHaemophilus ducreyi
Treponema pallidumClostridium species
Mycobacterium marinumPseudomonas aeruginosa
Corynebacterium diphtheriae

67. Specimen Collection
Swabs
1. Inform the patient.
2. No-touching technique: remove bandage with the forceps.
3. With the forceps take a sponge, dip it in the saline and wash
the surface of the wound or ulcers free from exudate.
4. Remove the swab from its covering and extend the tip of the
swab deep into the wound taking care not to touch the
adjacent skin margins.
5. Remove the stopper from the test tube with transport medium,
plunge the swab into the transport medium and replace the
stopper, if the wooden stick of the swab is too long, break off
the end over the rim of the test tube.
6. Apply new bandage.
7. Wash hands and fill-in the request form.

68. Wound Infection Pathogens and Commensals
Commensals bacteria
Coagulase negative Staph.Alpha haemolytic streptococci
Propionobacterium spp.Corynebacterium spp.
Bacillus spp.
Pathogenic bacteria
Streptococcus pyogenesPseudomonas aeruginosa
Staphylococcus aureusProteus spp
Enterococcus spp.E.coli
Clostridium perfringesKlebsiella spp
Fusobactrium sppMorganella
Peptostreptococcus sppProvidencia
Actinomyces israeliiMycobacterium tuberculosis
Nocardia spp.Bacteroid spp.

69. Specimen Processing
 Direct smears.
 Culture.
1. Enrichment and selective media included blood
agar, chocolate, MacConky, in addition to a tube
of Thioglycolate broth media is inoculated and
incubated for 24 hours in 37 C incubator.
2. In case of suspected anaerobic organisms
another blood agar plate is streaked and
incubated anaerobically for 48 hours.

70. Eye Culture

71. Infection of Eyes

72. CommensalsCommon pathogen
Staphylococcus epidermidisStreptococcus pyogenes
Lactobacillus sppPseudomonas aeruginosa
Propionibacterium sppChlamydia trachomatis
Staphylococcus aureusStreptococcus pneumoniae
Various EnterobacteriaceaeHaemophilus influenzae
Various streptococcus sppHaemophilus aegypticus
Occasion pseudomonas aeruginosaStaphylococcus aureus
Neisseria gonorrheae

73. Specimen Collection
1. Pull down the lower eyelid so that the lower conjunctival
fornix is exposed.
2. Swab the fornix without touching the rim of the eyelid with
the sterile cotton swab.
3. Place the swab immediately in a bacterial transport medium
or, if the specimen is brought to the laboratory immediately,
in a sterile test tube with 0.5 mL of buffered saline (pH 7).
Specimen Processing
1. Direct Visual Examination
 Specimen in which chlamydia is suspected can be stained
immediately with monoclonal antibody conjugated to
fluorescein for detection of elementary bodies or inclusions.
If a- canthomoeba or other amoebae are suspected, a direct
wet preparation should be examined for motile trophozoites.

74. 2. Culture
 the number of organisms recovered from cultures of certain eye
infection may be relatively low.
 blood and chocolate agar plates incubated under increased carbon
dioxide tension (5-10%).
 it maybe very helpful when any one eye is infected to culture both
eyes.
 If a potential pathogen grows in cultures of both infected and un
infected eye the organisms may not be causing the infection, now
ever if the organism only grows in culture from the infected eye, it
is most likely the causative.
3. Non Culture Methods
 ELISA and DFA staining are now available for detection of
Chlamydia trachomatis, an ELISA test is available also for detection
of Toxocara infection, finally single and multiplex polymerase chain
reaction assays are being developed for the diagnosis of viral and
Chlamydia keratoconjunctivities.

75. Specimen processing for conjunctiva
Because the constant washing action of the tears the number of
organisms recovered from cultures of certain eye infection may be
relatively low, so Conjunctival scrapings place directly onto media yield
the best results.

77. Genital Tract Infections

78. Commensals bacteriaPathogenic bacteria
coagulase negative StaphNeisseria gonorrheae
Corynebacterium spp.Group B Streptococci
E.coli and other coliformGardnerella vaginalis
Many species of anaerobicEnterococcus spp.
Certain anaerobes including
Actinomyces spp.
Haemophilus ducreyi
Treponema pallidum
Mycoplasma spp.
Enterobacteriaceae
Chlamydia trachomatis
Infections of Genital Tract

79. Specimen Processing
Direct Microscopic Examination
1. Uretheral discharge maybe examined by gram stain for the
presence of gram negative intracellular diplococci usually
indicative of gonorrhea in males.
2. Fluorescein conjugated monoclonal antibody reagents are
sensitive and specific for visualization of the inclusion of
Chlamydia trachomatis in cell culture or elementary bodies
in urethral and cervical specimens containing cells.
3. Direct microscopic examination of a wet preparation of
vaginal discharge provides the simplest rapid diagnostic test
for Trichomonas vaginalis.

80.  Budding cells and pseudohyphae of yeast can also be easily
identified in wet preparation by adding 10% KOH.
 Clue cells contain Gardenerella vaginalis adhering to the
epithelial cell can be demonstrated with wet preparation or
gram stain .
Culture
1. Streak two blood one Aerobic and the other
Anaerobicagar plates, one chocolate, MacConkey and
Sab agar plate.
2. Samples for isolation of gonococci maybe inoculated
directly to culture media as modified Thayer- Martin
media, New York City media.

82. Antibiotics used in prostatic culture
Gram negativeGram positive
CefuroximeCefuroxime
DoxycyclineDoxycycline
Co-trimoxazoleCo-trimoxazole
CiprofloxacinCiprofloxacin
MinocyclineMinocycline
TetracyclineTetracycline
OfloxacinOfloxacin
Erythromycin
Rifampicin

83. Stool Culture

84. Gastrointestinal Tract Infections

85. Commensals floraCommon pathogens
Enterobacteriaceae other than the
common pathogens
Helicobacter pylori
Bacteroides sppSalmonella spp.
Streptococcus sppE. coli O157:H7
LactobacilliStaph aureus
Pseudomonas spp.Campylobacter spp.
Coagulase negative staphylococciVibrio cholerae
BacteroidesYersinia enterocolitica
ClostridiumClostridium difficile
PeptostreptococcusShigella spp.
Bifidobacterium
Eubacterium.

86. Aim of the test
 Detect bacterial pathogenic organisms in the stool; only for
Salmonella spp. or Shigella spp.
Types of specimen
 Stool, rectal swab in fecal transport system or Duodenal or
sigmoid aspirate.
Criteria of specimen rejection
 specimen contaminated with urine, residual soap, or
disinfectants.
 Specimens received in grossly leaking transport containers,
Diapers, dry specimens.
 specimens submitted in fixative or additives.
Routine Stool culture

87. Specimen Processing
Wet Mount:
 fastest for detection of motile trophozoites of Entamoeba, Giardia
and other intestinal parasites.
Staining :
 don’t prefer making gram stain for stool samples..
Antigen detection:
 Enzyme immuno assays or latex agglutination can detect numerous
microorganisms
Culture:
1. examined For the presence of Salmonella, and Shigella spp.
2. One gram of stool is transported to tube of selenite F broth and a
loop is streak on XLD or SSA and incubated at 37ċ, after the
overnight incubation subculture from selenite F broth onto a fresh
plate of XLD or SSA.

88.  For un routine culture the physician must specify in the
request.
 Campylobacter spp. Can isolate on campy blood agar plate
media .
 Plates should be incubated in a microaerophilic atmosphere
at 42ċ and examined at 24-48 hours for suspicious colonies.
 E.coli O157H7 can be isolated on MacConky agar or Sorbitol
MacConky agar and incubated at 35-37ċfor 24 hours.
 Suspected colony must identified by using biochemical test as
API 20E and E.coli O157H7 antiserum for non Sorbitol
fermenters bacteria.

89.  Stool from patient suspected to have Cholera is streaked onto the
surface of TCBS plate and about one gram is incubate into a tube
containing alkaline peptone water.
 incubate at 37 oC, after 6-8 hours make a subculture from the
alkaline peptone water onto the surface of a new plate of TCBS
incubate at 37ċ for 24 hours.
 Colony that growth will show yellow color according to the
fermentation of Sucrose sugar, suspected colony must identified by
using biochemical tests as API system, antiserum for Vibrio are
available and must used to confirm the diagnosis.

90. Specimen processing

91. Shigella and Coliforms on (HEA)
Salmonella on (XLD)
Salmonella and Shigella on (SSA)

92. Salmonella, Shigella, Vibrio, and Campylobacter
GentamicinAmpicillin
CefotaximeCo-trimoxazole
Erythromycin
(for Vibrio and Campylobacter.)
Doxycycline
Ciprofloxacin