Module 5:

Specimen
Processing
Learning Objectives (1)

At the end of this session, participants should be able to:

 List normal flora organisms that can be found in

different body sites
 Describe how normal flora organisms can
complicate sample collection & interpretation of
culture results
 Define opportunistic infection
 List organisms that are common causes of
opportunistic infections by specimen type
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Learning Objectives (2)

 Define nosocomial infection

 List organisms that are common causes of
nosocomial infections
 List pathogens most likely to be isolated as the
cause of disease from the 8 most common
specimens received in the clinical microbiology
laboratory
 Describe methods (media, temperatures &
atmospheres) used for the primary culture of the 8
most common specimens

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Content Overview

 Normal flora of various body sites

 Opportunistic & Nosocomial infections
 Organisms found in the 8 most common specimens
- Blood
- Cerebrospinal fluid (CSF) & other body fluids
- Stool
- Genital
- Wound, pus, aspirate, tissue
- Sputum
- Throat
- Urine
- Eye, ear (specific to Ethiopia)
 Processing of the 8 most common specimens
 Temperature & atmospheres of incubation
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Normal Flora
Normal Flora (1)

 Normal flora (NF): bacteria & fungi that are

permanent residents of body sites
 The number & types of NF vary from site to site
 Play a role in both the maintenance of health & in
causing diseases
 Cause disease in immunocompromised or in normally
sterile sites
 Act as a protective host defense mechanism
 May serve a nutritional function
 Can complicate interpretation of culture results
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Normal Flora (2)

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Opportunistic Infections

 An infection caused by an organism that

does not cause disease in a healthy host
 Opportunistic pathogen – Examples:
 Oral infections - Candida albicans
 Pneumonia - Legionella pneumophila

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Nosocomial Infections

 An infection that occurs as a result of treatment in

a hospital or a healthcare service facility
 Many nosocomial infections are due to organisms
that are resistant to multiple antibiotics
 Common nosocomial pathogens
 E. coli & other enteric organisms
 Staphylococcus aureus
 Pseudomonas aeruginosa
 Acinetobacter

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Specimen Processing

CARE GIVER

PATIENT

LABORATORY

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Blood
Blood Cultures

 Clinical condition – Septicemia/ Endocarditis

 Terms:
 Transient – bacteremia, no clinical consequence
 Continuous – organisms continually present &
multiplying in the blood stream - septicemia
 Outcome of septicemia
- Sepsis
- Shock
- 20-40% mortality with treatment
- 100% mortality if untreated
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Blood Collection

The number of blood cultures to draw, when to draw

them, how to prepare the collection site, & how
much blood to draw are complicated issues that are
different in varying patient populations & circumstances.
There are however, some general guidelines that will be
presented here.
There are many good resources with detailed
instructions for specimen collection. One very good
reference is:
Clinical Microbiology Procedures Handbook, ASM
Press
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Collection of Blood (1)

 Number:
 2-3 separate collections (from different sites)
 Aerobic & anaerobic bottle for each collection

 Volume: Recommended ratio of blood drawn to

culture media is 1:5 – 1:10
 Adults 30 – 40 ml per 2 draws – volume is important due to
low numbers of circulating bacteria
 Children-amount depends on weight of child

 Avoid drawing blood from IV catheters

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Collection of Blood (2)

 Timing
 Prior to/during a fever spike
 BEFORE antibiotics if possible

 Venipuncture site preparation

 Vigorous scrub with alcohol & allow to dry
 Swab or wipe concentric circles of tincture of iodine or
chlorhexidine, moving outward from the center of the site.
Reswipe with alcohol to remove the iodine. DO NOT
repalpate site after disinfection.

 Blood culture bottle preparation

 Disinfect septum with alcohol & allow to dry
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Common Isolates from Blood Cultures: Gram-positive

 Staphylococcus aureus
 Coagulase-negative Staphylococcus
 Viridans group streptocococci
 Streptococcus pneumoniae
 Streptococcus pyogenes
 Enterococcus faecalis

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Common Isolates from Blood Cultures: Gram-negative

 Enterobacteriaceae
 E. coli is one of the most common blood culture
isolates
 Neisseria meningitidis
 Pseudomonas aeruginosa
 Haemophilus influenzae

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Blood Culture Contaminants – A Big Problem

Contamination can occur at the time of collection

or in the laboratory while subbing the blood
culture bottle.
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Common Contaminants

Normal skin flora

 Coagulase negative staphylococci **
 Coryneforms (diphtheroids) **

Environmental
 Bacillus sp.

**Unless the same strain is present in multiple sets

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Cerebrospinal Fluid (CSF)
CSF Culture

 Clinical condition - Meningitis

 Pre-Hib vaccine, most common in young children
 May occur as epidemics in small groups (primarily
meningococcus)
 Symptoms
 Fever, vomiting, headache, drowsiness & seizures

 Rapid progression – medical emergency

 Neisseria meningitidis can affect several organs-
coma & death
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Causes of Acute Bacterial Meningitis

 Neonates – 2 months
 E. coli
 Streptococcus agalactiae - Group B Streptococcus
 Listeria monocytogenes

 2 months – 2 years
 Haemophilus influenzae type B (if no vaccination)
 Neisseria meningitidis
 Streptococcus pneumoniae

 > 2 years - adults

 Neisseria meningitidis
 Streptococcus pneumoniae
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CSF Specimen Collection

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CSF Processing

 CSF – collected aseptically

 Delivered to the laboratory immediately in sterile
tubes at room temperature
 Ideal to collect at least 4 sequential tubes
 Tube 1 – Chemistry – protein & glucose
 Tube 2 – Microbiology – bacterial & fungal
 Tube 3 – AFB & other special tests
 Tube 4 – Hematology – cell count

 NOTE
 Do not refrigerate CSF
 Critical specimen—process & report Gram stain
results immediately
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CSF Analysis

Etiology Pressure Cells Protein Glucose

(per mm H2O) (per mm3) (mg/100cc) (mg/100cc)

Normal <200 0-5 lymphs <45 >40 or 2/3

CSF
0 polys sugar

Bacterial Increased 200-500 >100 <40

(mostly polys)

Viral Sl. increase 100-700 Sl. increase Normal

(mostly lymphs)

Chronic Increased 25-500 >100 <40

(mostly lymphs)
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Page 27
Meningitis in the Immunocompromised Host

 Two important causes

 Listeriamonocytogenes – also important cause in
pregnancy
 Cryptococcus neoformans – fungus

Perform India Ink

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Stool
Routine Stool Culture

 There are many different enteric pathogens

 A routine stool culture should be able to detect
the following common enteric pathogens
 Salmonella
 Shigella
 Other pathogens, such as Vibrio cholerae, E.
coli 0157:H7, & Campylobacter require special
media & must be specifically requested for
isolation
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Page 31
Genital
Genital Cultures: Clinical Conditions

Genital cultures are primarily performed to

diagnose certain bacterial Sexually Transmitted
Infections (STIs) or vaginal infections.

Culture is also performed to determine if

pregnant women are carriers of Group B
streptococci

FEMALES: Urethritis & Cervicitis, Vaginitis,

Bacterial Vaginosis (BV)
MALES: Urethritis
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Culture for STIs - Bacterial Pathogens

 Neisseria gonorrhoeae (GC) - urethritis in

male & female, cervicitis in females
 Chlamydia trachomatis – non-gonococcal
urethritis/cervicitis – requires cell culture,
often a diagnosis of exclusion

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Organisms Causing Vaginitis

 Candida albicans - vaginitis

 Trichomonas vaginalis – vaginitis
 Overgrowth of Gardnerella vaginalis & anaerobic
vaginal flora – BV

1. Vaginal wet mounts are often done to

diagnose these infections
2. Cultures can be done for C. albicans & T.
vaginalis
3. Gram stains are useful for C. albicans & BV

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Genital Tract: Anatomy & Specimen Collection

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Processing of Genital Specimens for GC

GC CULTURE – Thayer-Martin Media

 Females - cervical, urethral & anal swabs
 Males - urethral & anal swabs
 These must be immediately placed in transport media
(Amies with charcoal) & delivered to the laboratory
 DO NOT REFRIGERATE

GRAM STAINS:
 Female cervical specimens – Not done, low specificity &
sensitivity
 Male urethral – Yes, high specificity & sensitivity
 Anal - ?

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Genital Specimens

 Vaginitis
 High vaginal swab (HVS) in sterile saline for wet mount
 HVS in transport media for culture – Candida albicans or
other yeast

 Bacterial vaginosis
 HVS in sterile saline for wet mount or Gram stain. Culture is
not done.

 Group B Streptococci carriage-done at 35 – 37

weeks gestation
 Vaginal/anal swab in transport media
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Bacterial Vaginosis

 Alteration of normal vaginal flora (Lactobacilli replaced

with G. vaginalis & anaerobes)
 Diagnosis:
 “Clue Cells” seen on wet mount - epithelial cells covered with rod-
shaped bacteria – G. vaginalis
 Gram stain:
- Lactobacilli are either absent or few in number
- many “clue cells present” – Epis covered in gram-variable rods
- many gram-negative anaerobes - Mobiluncus species & Bacteroides species
predominate
 Whiff Test using 10% KOH (fishy odor elicited by the addition of a
drop of 10% KOH to the discharge on a slide)
 A pH of > 4.5
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Example of “Clue Cells” in Wet Mount

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Example of “Clue Cells” in Gram Stain

 Note gram-variable rods

attached to squamous
epithelial cells
 Not gram-negative rods -
anaerobes

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Page 42
Pus, Aspirate, Tissue
Pus, Aspirate, Tissue (1)

 Clinical condition
WOUNDS, or ABCESSES, from simple trauma to
burns, post-operative infections

 Pathogens
A wide variety of aerobes & anaerobes such as
S.aureus, S.pyogenes, Enterobacteriaceae,
Clostridia & Bacteroides sp.

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Pus, Aspirate, Tissue (2)

 Specimens
 Wound swabs - care to avoid contamination
with normal skin flora
 Surgical aspirates & biopsies

*Swabs placed are in transport medium,

aspirates & biopsies are placed in sterile
containers
All specimens should be delivered to the
laboratory as soon as possible
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Page 46
Sputum
Lower Respiratory Tract

 Clinical conditions

Pneumonia, Bronchitis, lung abscess

 Specimen
 Expectorated sputum, induced sputum, tracheal
aspirations, endotracheal tube secretions
 Collect in a sterile container
 Delivered to the laboratory (refrigerate if delay)

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Lower Respiratory Tract Pathogens

The organisms that cause pneumonia can differ

significantly depending on:
 Community acquired vs. hospital acquired
 Immune status of patient
 Age & demographics of patient
 Organisms considered part of the “normal oral
flora” & should be reported as such:
Viridans streptococci, non-pathogenic Neisseria spp.,
Coagulase-negative staphylococcus (CNS), small
numbers of yeast, diptheroids
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Common Lower Respiratory Tract Pathogens (1)

Community-acquired
 S. pneumoniae*– most common
 Mycoplasma pneumoniae **– young adults, closed
populations
 Chlamydia pneumoniae **
 S. aureus* – following viral infections
 H. influenzae* in children & smokers.
 K. pneumoniae – alcoholics & others with chronic diseases
 Legionella species**

*These organisms can also be found as normal oral flora. Report

only if predominating organism.
**These organisms require special culture techniques & will not
grow on a routine sputum culture.
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Common Lower Respiratory Tract Pathogens (2)

 Hospital-acquired
 Enteric gram-negative rods
 S. aureus
 Pseudomonas aeruginosa – other non-fermenters
 Streptococcus pneumoniae
 H. influenzae
 Legionella species

ICU patients & patients with a prolonged hospital course

often become colonized in the oropharynx with gram-
negative rods. Report the isolation of these organisms only
if present in significant numbers.
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Sputum Gram Stain

Gram stain is used to assess acceptability of

sputum specimen for culture:
 Scan under low power (LPF)- several
representative fields
 Count & record polymorphonuclear
leukocytes (PMN’s) & squamous epithelial
cells (SEC’s)
> 10 SEC/LPF---------Reject, Poor quality,
suggestive of saliva
 < 10 SEC/LPF----------Culture
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Gram Stain: Sputum (1)

Unacceptable Specimen – 100x

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Gram Stain: Sputum (2)

Unacceptable Specimen – 1000X

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Gram Stain: Sputum (3)

Acceptable Specimen – 100X

Page  55
Page 55
Gram Stain: Sputum (4)

Acceptable Specimen - 1000X

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Page 57
Eye & Ear
Eye Culture

 Clinical Conditions:
 Conjuctivitis, keratitis, cellulitis, & other anatomical areas of eyes -
often respiratory organisms are the causative agent
 Common pathogens: Haemophilus influenza, Staphylococcus aureus,
Streptococcus pneumoniae, Group A Streptococcus, Moraxella sp., &
Pseudomonas aeruginosa. Also Neisseria gonorrhoeae.

 Interpret culture by taking into consideration: pure culture,

predominant organism, know pathogen, association with
PMN’s, & quantity.
 Culture on Blood agar plate & Chocolate in CO2 for 48-72
hours
 Routine culture will not recover other pathogens such as
virus, chlamydia, mycobacteria, acanthamoeba, & anaerobes
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Ear Culture

Clinical conditions
 Otis media - an infection of the middle ear
 Common pathogens: S. pneumoniae, H. influenza, & Moraxella
catarrhalis
 Otis externa – an infection of the external ear canal
 Common pathogens: P. aeruginosa (swimmer’s ear), S. aureus, &
Group A Streptococcus
Poorly collected specimens may be contaminated with skin flora:
Coagulase-negative Staphylococcus & Corynebacterium sp.
 Interpret culture by taking into consideration: pure culture,
predominant organism, know pathogen, association with PMN’s
& quantity
 Culture on blood agar plate, Chocolate, in CO2 for 48-72 hrs &
MacConkey
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Throat
Throat Culture

 Clinical condition – Pharyngitis, tonsillitis

 Approximately 80% caused by viruses
 Most of the rest of the cases are caused by a single
bacterium
Streptococcus pyogenes – Group A Strep
 Uncommon causes require special culture
techniques – physician MUST notify the laboratory
 Corynebacterium diphtheriae
 Neisseria gonorrhoeae

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Routine Throat Culture

 SPECIMEN:
Throat swab—
sample the inflamed area

 TRANSPORT:
In Amies/Stuart transport media or…
This is the only specimen that can be
transported to the laboratory as a dry swab

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Page 64
Urine
Urinary Tract: Anatomy

The majority of
urinary tract
infections (UTI)
occur in women

Clinical Conditions
Lower UTI: Infection of bladder - Cystitis
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Upper UTI: Infection of Kidney - pyelonephritis
Urinary Tract Pathogens & Specimens

Pathogens
E. coli, Proteus sp, K. pneumoniae, Enterococci, S.
aureus, S. saprophyticus

Specimens
 Clean catch mid-stream urine
 Catheter - indwelling, straight
 Cystoscopy

*In sterile container—refrigerate if delay

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Urinalysis

 Urinalysis – performed in other sections of the

Laboratory - can be useful in interpreting urine
culture results
 Culture — Urine is a sterile body fluid
 Female specimens are easily contaminated with normal
flora of the perineum or vagina
 Males – specimens usually less contaminated
 Quantitative culture done, higher bacterial counts have a
strong correlation with urinary tract infection
 Specific volume cultured to provide colony forming units
(cfu)/ml
Interpretation is based on method of collection &
clinical condition
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Page 69
Temperatures & Atmospheres of Incubation (1)

Temperature: 35 – 37°C
Atmospheres: Organisms differ in the
requirements for oxygen (O2) & carbon
dioxide (CO2)
 Obligate aerobe: requires O2 for growth –
examples: Pseudomonas & Mycobacterium.
 Obligate anaerobe: will not grow in the presence
of even small amounts of O2 – examples:
Bacteroides & Clostridium species
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Temperatures & Atmospheres of Incubation (2)

 Facultative: will grow in the presence or absence

of O2 – examples: S. aureus or E. coli
 Microaerophile: Requires reduced (about 6%) for
growth – example: Campylobacter species
 Capnophile: Requires increased CO2 (5 – 10%)
for growth – example: N. gonorrhoeae, meningitidis
& Haemophilus influenzae

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Specimen Processing Summary
Specimen, Media, Purpose, & Incubation (1)

SPECIMEN MEDIA PURPOSE INCUBATION

Positive blood Sheep Blood Agar Gram Positive orgs 35oC in CO2 for
culture MacConkey Gram Negative orgs 72 hours
Chocolate Yeast
CSF Sheep Blood Agar S. pneumoniae 35oC in CO2 for
Group B Strep 72 hours
E. Coli
Chocolate H. influenzae
N. meningitidis
Throat Sheep Blood Agar Beta Strep, Group A 35oC in CO2 for
48 hours
Sputum Sheep Blood Agar Gram Positive orgs 35oC in CO2 for
yeast 48 hours
MacConkey Gram Negative orgs
Chocolate H. Influenzae
Pus/tissue/ Sheep Blood Agar Gram Positive orgs 35oC in CO2 for
Aspirate MacConkey Gram Negative orgs 48 hours
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Specimen, Media, Purpose, & Incubation (2)

SPECIMEN MEDIA PURPOSE INCUBATION

Urine Sheep Blood Agar Gram Positive orgs 35oC in air for
MacConkey Gram Negative orgs 24 hours
or
CLED Gram Positive orgs
Gram Negative orgs
Stool Sheep Blood Agar Enteric flora 35oC in air for
Routine Culture MacConkey Salmonella / 48 hours
HEK (preferred) or Shigella)
XLD
Campy Agar Campylobacter 42oC
Microaerophilic
for 72 hours
Stool
Cholera Culture Add: APW & TCBS Vibrio cholera 35o C in air for
48 hours

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Specimen, Media, Purpose, & Incubation (3)

SPECIMEN MEDIA PURPOSE INCUBATION

Genital Male Modified Thayer GC 35oC in CO2 for
Urethral/anal Martin 72 hours

Genital Female
Cervix/anal Modified Thayer GC 35oC in CO2 for
Martin 72 hours

Vaginal/anal Sheep Blood Agar Strep. Group B 35oC in CO2 for

(35-37 wks preg.) Enrichment Broth 48 hours

Vaginal swab Sheep Blood Agar Yeast 35oC in CO2 for

or Sabouraud 72 hours

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Summary

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