Specimen Processing.ppt

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 organisms from the normal flora can
complicate sample collection & also affect the
interpretation of culture results
Define opportunistic infection
List organisms that are common causes of
opportunistic infections by specimen type

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

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
 Processing of the 8 most common specimens
 Temperature & atmospheres of incubation

Normal Flora (1)
Normal flora (NF): bacteria & fungi that are
permanent residents of body sites
 The number & types of NF vary in different sites
 Play a role in the maintenance of health as a
protective host defense mechanism, and may serve
a nutritional function
 Can cause disease in immuno-compromised hosts,
or when introduced into normally sterile sites
 When isolated in culture they can make the
interpretation of results difficult.

Opportunistic Infections
 An infection caused by an organism that
does not cause disease in a healthy host
 Opportunistic pathogen – Examples:
 Oral infections - Candida albicans
 Prosthetic valve endocarditis- coagulase
negative staphylococci

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

CARE GIVER
LABORATORY
PATIENT
Specimen Processing

Blood Culture
Clinical condition – Bacteraemia
 Transient –no clinical consequence
 Intermittent- from an infected site-abscess
 Continuous – organisms continually present &
multiplying in the blood stream -
 Outcome of bactaeremia
- Sepsis
- Shock
- 20-40% mortality with treatment
- 100% mortality if untreated

Common Pathogens
Gram-positive
Staphylococcus aureus
Coagulase-negative
Staphylococcus
Viridans group
streptocococci
Streptococcus
pneumoniae
Streptococcus pyogenes
Enterococcus faecalis
Gram-negative
Enterobacteriaceae
 E. coli is one of the most
common blood culture
isolates
Neisseria meningitidis
Pseudomonas
aeruginosa
Haemophilus influenzae

Blood Collection (1)
The number of blood cultures to draw, when to draw
them, & how much blood to draw varies among
patient populations & clinical situations.
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

Blood Collection (2)
Avoid drawing blood from IV catheters
Timing
 Prior to/during a fever spike
 BEFORE antibiotics if possible
Venipuncture site preparation
 Cleanse the site 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

Blood Culture Contaminants
Common Contaminants
Normal skin flora
 Coagulase negative
staphylococci *
 Coryneforms (diphtheroids) *
Environmental
 Bacillus sp.
*Unless the same strain is
present in multiple sets
Contamination can occur at the time of
collection or in the laboratory while
subculturing the blood culture bottle

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

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

Meningitis in the Immunocompromised Host
Two important causes
 Listeria monocytogenes – also important cause in
pregnancy
 Cryptococcus neoformans – fungus
India Ink
Preparation

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

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 PMN’s sugar
Bacterial Increased 200-500 >100 <40
(mostly PMN’s)
Viral Sl. increase 100-700 Sl. increase Normal
(mostly lymphs)
Chronic Increased 25-500 >100 <40
(mostly lymphs)

Stool Culture
 Clinical conditions – watery diarrhea, dysentery,
typhoid fever
 Inflammatory Diseases
- Salmonella
- Shigella
- Campylobacter
 Toxin-mediated Diseases
- Vibrio cholerae
- Enterohemorrhagic E. coli (O157:H7)

Stool Collection
Specimen types:
 Stool from patients in clean containers
 Fecal or rectal swabs that are kept moist
 Filter paper, gauze, or cotton soaked in liquid stool
(keep moist inside a plastic bag)
 Specimens in transport medium (Cary-Blair)
The recovery of enteric pathogens is dependent on the
organism surviving transport
Many enteric pathogens will die if not preserved in
transport
medium

Genital Culture
Genital cultures are performed to diagnose:
 Sexually Transmitted Infections (STIs)
Urethritis &Cervicitis in females; Urethritis in males
Vaginal infections:
Vaginitis & Bacterial Vaginosis ( BV)
Carriage of Group B streptococci in pregnant
women

Common Genital Tract 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
Candida albicans - vaginitis
Trichomonas vaginalis – vaginitis
Overgrowth of Gardnerella vaginalis & anaerobic
vaginal flora – BV

Genital Tract Specimen Collection

Processing of Genital Specimens (1)
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 swab– Yes, high specificity & sensitivity

Processing of Genital Specimens (2)
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

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

Examples of “Clue Cells”
Clue Cells in Wet Mount
Clue Cells in Gram Stain
 Note gram-variable rods attached
to squamous epithelial cells
 Not gram-negative rods -
anaerobes

Pus, Aspirate, Tissue Culture
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.

Pus, Aspirate, Tissue Collection
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

Lower Respiratory Tract
Clinical conditions
Pneumonia, Bronchitis, lung abscess
The organisms that cause pneumonia can differ
significantly depending on:
 Community acquired vs. hospital acquired
 Immune status of patient
 Age & demographics of patient
Specimen
 Expectorated sputum, induced sputum, tracheal aspirations,
endotracheal tube secretions
 Collect in a sterile container
 Delivered to the laboratory (refrigerate if delay)

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.

Hospital-acquired
 Enteric gram-negative rods
 S. aureus
 Pseudomonas aeruginosa – other non-fermenters
 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.
Common Lower Respiratory Tract Pathogens (2)

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

Gram Stain: Sputum (1)
Unacceptable Specimen – 100x

Unacceptable Specimen – 1000X

Page  50
Acceptable Specimen – 100X

Acceptable Specimen - 1000X

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

Throat Specimen Collection
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

Urinary Tract (1)
Clinical Conditions
Lower UTI: Infection of bladder - Cystitis
Upper UTI: Infection of Kidney - pyelonephritis
The majority of
urinary tract
infections (UTI)
occur in women

Urinary Tract (2)
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

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

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

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

SPECIMEN MEDIA PURPOSE INCUBATION
Positive blood
culture
Sheep Blood Agar
MacConkey
Chocolate
Gram Positive orgs
Gram Negative orgs
Yeast
35oC in CO2 for
72 hours
CSF Sheep Blood Agar
Chocolate
S. pneumoniae
Group B Strep
E. Coli
H. influenzae
N. meningitidis
35oC in CO2 for
72 hours
Throat Sheep Blood Agar Beta Strep, Group A 35oC in CO2 for
48 hours
Sputum Sheep Blood Agar
MacConkey
Chocolate
Gram Positive orgs
yeast
Gram Negative orgs
H. Influenzae
35oC in CO2 for
48 hours
Pus/tissue/
Aspirate
Sheep Blood Agar
MacConkey
Gram Positive orgs
Gram Negative orgs
35oC in CO2 for
48 hours
Specimen, Media, Purpose, & Incubation (1)

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

Genital Male
Urethral/anal
Modified Thayer
Martin
GC 35oC in CO2 for
72 hours
Genital Female
Cervix/anal Modified Thayer
Martin
GC 35oC in CO2 for
72 hours
Vaginal/anal
(35-37 wks preg.)
Sheep Blood Agar
Enrichment Broth
Strep. Group B 35oC in CO2 for
48 hours
Vaginal swab Sheep Blood Agar
or Sabouraud
Yeast 35oC in CO2 for
72 hours

Specimen Processing.ppt

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