SAMPLE

PROCESSING •
•
KEKI(96)
KHADHIJA(97)
(PUS •
•
KHUSHI S(98)
KHUSHI VINOD (99)

,URINE,BODY • KOSURU SANSITHA LAASYA(100)

FLUIDS )
WHAT IS A SAMPLE?
• The term specimen is very commonly used in the laboratory to
indicate a sample taken from the human body.
• IMPORTANCE OF SPECIMEN COLLECTION
• For the purpose of correct diagnosis.
• Therapeutic decisions rely, in part, on accuracy of test results.
Types of sample collected
Skin and soft tissue infection – Pus
Urinary tract infection – Urine
BODY FLUIDS
CSF
Synovial fluid
Gastric Aspirate
Pleural Fluid
Pericardial Fluid
Vaginal Secretions
Peritoneal Fluid
Aspirate from inner Ear etc
(we will be focussing on pus , urine and body fluids)
General principles while collecting samples
• Standard precautions should be followed while collecting and
handling the sample like Hand hygiene , PPE ,Biomedical waste
Disinfection of patient care items
• Culture specimen should be collected prior to
ANTIBIOTIC administration
• CONTAMINATION with indigenous flora should be avoided especially
like urine and blood culture specimen
• Specimen should be collected in sterile ,tightly sealed ,leakproof ,
Wide mouth, screw capped container
• Labeling : All specimen should be appropriately labelled with
name, age , gender ,treating physician ,diagnosis ,antibiotic
history, Type of specimen and desired investigation name
• If anaerobic culture is requested, proper anaerobic collection
container with media should be used
Specimen Transport
• The specimen should reach the laboratory for further process as soon
as possible after the collection
• For most of the specimen, transport time should not exceed more
than two hours however there are some exceptions
• Specimen that require an immediate transport (<15 minutes ) -such
has CSF and body fluids ,tissue specimen ,bone specimen .
• Urine added with preservative is acceptable upto 24 hours ,otherwise
should be transported within 2 hours
• Stool specimen should be transported within 1 hour
Process of sample collection
• Completely fill requisition form
• Properly labeled and leakproof container
• Adequate volume of fluid
• Collect sample at optimum time and from actual site of infection
• Instruction to collect the sample should to given in local language
• Proper transportation
Rejection criteria
• Mismatch identification
• Insufficient quantity
• Inappropriate container
• Contamination suspected
• Inappropriate transportation temperature
• Excessive delay in transport
• Dry specimen
• Inappropriate transport media
PUS
A thick, opaque, usually yellowish-white fluid matter
that is formed as a part of an inflammatory response
typically associated with an infection and is composed
of exudate chiefly containing dead white blood cells (
neutrophils), tissue debris and pathogenic
microorganisms (such as bacteria)
 Ways to collect pus samples:

Collection -
-
Aspiration
Swab collection
and delivery - Abscess drainage
Indications:
• Localized Infections:
Abscesses
Cellulitis
• Wound Infections:
SURGICAL SITE INFECTIONS
ANAEROBIC INFECTIONS
Traumatic wounds
• Soft Tissue Infections:
Furuncles (boils) and carbuncles
Cellulitis
• Joint Infections:
Septic arthritis
• Visceral Abscess:
Liver abscess, lung abscess, etc.
• Deep Tissue Infections:
Fasciitis
Osteomyelitis

• Dental Infections:
Dental abscess

• Systemic Infections:
Sepsis
Pyomyositis

• Chronic Infections:
Chronic wounds
Chronic sinusitis

• Device-Related Infections:
Infections related to indwelling catheters, prosthetic devices, or
implants
Why understanding normal flora is
important?
Helps distinguish between normal flora and pathogens causing infections.

Facilitates accurate interpretation of laboratory culture results from pus samples.

Prevents Overdiagnosis and Reduces the risk of incorrectly identifying normal flora as infectious agents.

Informs healthcare providers about which antibiotics are likely to be effective against the identified pathogens.

Aids in the responsible use of antibiotics, minimizing the risk of antibiotic resistance.

Helps identify individuals at higher risk of certain infections based on their normal flora.
Likely pathogens:
• Gram-Positive Bacteria: • Gram-Negative Bacteria: • Fungi:
Staphylococcus aureus Pseudomonas aeruginosa Histoplasma

Streptococcus pyogenes Escherichia coli Candida

Proteus species Fungi causing mycetoma
Enterococcus species
Klebsiella species
Anaerobic streptococci
Bacteroides species • Parasites:
Other streptococci
Acinetobacter species
Clostridium perfringens and other clostridia Entamoeba histolytica (in pus
Other enteric bacilli aspirated from an amoebic
Actinomycetes liver abscess)
Mycobacterium tuberculosis

• Viruses:
Pox viruses
Herpes viruses
Laboratory examination of Pus sample:
appearance of the specimen:
presence or absence of sulfur granules (needed only for the suspected cases
of mycetoma or actinomycosis, when requested).
• If pus swab is sent:
• Only one aerobic pus swab: Inoculate the culture media first before using
the swab to make smears for Gram staining
• If swabs (one anaerobic and two aerobic) are submitted for culture, use
the second swab to make gram stain
• If tissue sample is submitted: make a Gram stain from ground tissue.
• If pus aspirate is sent, place one drop of pus onto a clean microscope slide using a
sterile pipette. Spread this using a sterile loop to make a thin smear for Gram
staining.
Preparation of the Smear:

Collect Label Slide: Prepare Spread Air-Dry: Fix the Stain

Sample: Slide: Evenly: Smear: (Optional):
Use a sterile Label a glass Place a drop of Mix pus and Let the smear Pass the slide Optionally, use
swab or loop slide with sterile saline saline, air-dry through a a stain like
to collect pus patient details on the slide. spreading it completely. flame a few Gram stain for
from the and sample Transfer a evenly on the times to fix the better
infected area. source. small amount slide. smear. visibility.
of pus into the
drop.
Gram staining:

Examine the smear for the presence of bacteria and pus cells (PMNs) using 100x objective
lens and look especially for:

• Gram-negative rods (possible pathogens are E.coli, Klebsiella

pneumoniae, Pseudomonas aeruginosa, Proteus or Bacteroides species)
• Gram-positive cocci in pairs, chains or clusters (possible pathogens
are Staphylococcus aureus, Streptococcus pyogenes, anaerobic streptococci
or enterococci).
• Gram positive large rods with square ends (possible pathogens are Clostridium
perfringens or Bacillus anthracis)
• In the case of anaerobic infections large number of pleomorphic bacteria
(streptococci, Gram positive and Gram negative rods of various sizes and
fusiform bacteria) may be seen. Sometimes, Gram-positive yeast cells
with pseudohyphae may be seen, which can be Candida albicans.
Pus culture
Culture Media:
Wound specimens collected on aerobic swabs or pus aspirate should be
plated on to the following media:
• Sheep blood agar (to isolate S. aureus and Streptococcus pyogenes or other
streptococci)
• MacConkey agar (to isolate Gram-negative rods)

Incubation Condition:
• Temperature: 35ºC -37ºC
• Atmosphere: Blood Agar plate in carbon dioxide enriched atmosphere (e.g. 5%
CO2 incubator or in a candle jar) and MacConkey agar plate in ambient air
(normal incubator)
• Time: Up to 48 hours (observe the plate after 24 hours of incubation, if growth
is seen, do further processing, if not, reincubate for additional 24 hours.)
Quantitation and its importance:
• Assesses Infection Severity:
• Quantification helps gauge the extent of the infection.
• Guides Treatment:
• Aids in determining the appropriate treatment course.
• Monitors Treatment Effectiveness:
• Allows tracking of how well the treatment is working.
• Informs Clinical Decisions:
• Provides crucial data for healthcare providers to make informed decisions.
• Standardizes Reporting:
• Follows established lab protocols, ensuring consistent and comparable results.
• Clinical Context:
• Results are interpreted in the context of the patient's overall clinical condition.
Examination and reporting:
Blood Agar Plate:
• Beta-Hemolysis: Look for hemolysis. S. aureus and S. pyogenes give beta-hemolysis. Some S. aureus may not show
hemolysis.
• Colony Morphology: S. aureus - yellow to cream or white, slightly raised, easily emulsified. S. pyogenes - small, colorless,
dry, shiny, or mucoid. Enterococci - non-hemolytic.
Catalase Test:
• Staphylococcus is catalase-positive, Streptococcus is catalase-negative.
Coagulase Test:
• Identify suspected S. aureus colonies.
• To differentiate coagulase-negative Staphylococci from S. aureus.
MacConkey Agar Plate:
• Lactose Fermenters: Look for pink colonies. E.g., Escherichia coli, Klebsiella spp, Enterobacter spp.
• Non-Lactose Fermenters: Look for pale colonies. E.g., Pseudomonas aeruginosa, Acinetobacter spp, Proteus spp.
Biochemical Tests for Identification:

• Citrate Utilization Test: Determines the ability to use citrate as a sole carbon source.
• Triple Sugar Iron (TSI) Agar Test: Assesses sugar fermentation and gas production.
• Sulphite-Indole–Motility (SIM) Test: Detects indole production, sulfur reduction, and
motility.
• Urease Test: Evaluates the ability to hydrolyze urea.

Antibiotic susceptibility
testing:
• For Streptococcus pyogenes and Enterococci, antimicrobial sensitivity testing should be
done in MHA supplemented with sheep blood.
• For S.aureus and other gram-negative bacilli, Mueller-Hinton Agar (MHA) is used.
• The selection of the antibiotics panel depends on the isolated organism. Unless
indicated routinely used (or first line), antibiotics should be used.
• If the patient is in an intensive care unit (SICU, PICU, NICU) or is receiving particular
antibiotic, or the isolate is resistant to first-line antibiotics, sensitivity testing should
include requested antibiotics and/or second-line antibiotics.
In case you cannot isolate pathogen:

Clinical Assessment:
• Rely on patient symptoms, history, and examination.
Review Lab Procedures:
• Ensure proper sample handling and processing.
Repeat Sampling:
• Consider re-sampling if the initial sample is inadequate.
Advanced Tests:
• Explore advanced diagnostic tests like PCR or serological assays.
Empirical Treatment:
• Initiate treatment based on the most likely pathogens.
Case study:
A 45-year-old patient presents with a high-grade fever, chills, and a rapidly spreading area of cellulitis on the lower
leg. The patient reports a recent injury while hiking. On examination, there are signs of systemic toxicity, and the
affected area is warm, erythematous, and tender. What is the most appropriate next step in the management of this
patient?

A) Prescribe oral antibiotics

B) Obtain a pus sample for analysis
C) Administer intravenous pain medication
Urine sample

Purpose of collecting urine sample

• Urine sample is collected for the diagnosis
and treatment of systemic and specifically
urogenital infections.
• Under microbiology, it is collected to identify
microorganisms via culture and to perform
antimicrobial susceptibility tests.
METHODS OF COLLECTION
• NON-INVASIVE METHOD
• Clean catch midstream urine
• INVASIVE METHOD
• Suprapubic aspiration
• Straight catheter
• Foley's catheter
 Rejection of Sample Unsuitable for
Processing

Some of the samples can rejected by the lab for various

reasons. Some of the following are:
• Bed pan or catheter bag or pooled 24 collection urine.
• If the specimen is unlabeled or improperly labeled.
• If the container is contaminated
Direct Examination
• Gram staining: limited to pyelonephritis and
invasive UTI.
• Wet mount examination: demonstrate pus
cells urine
• Leukocyte esterase test: detects leukocyte
esterases secreted by pus cells in urine
• Nitrate reduction test (Griess test): bacteria
like E. coli that reduce nitrate give a positive
result
 • CSF is a clear, colourless fluid present in the
subarachnoid space surrounding spinal cord
and brain.

• Collection of CSF :
CSF • It is done by performing a lumbar puncture.
A lumbar puncture is the diagnostic
procedure for the diagnosis of meningitis,
subarachnoid haemorrhage and certain
neurological disorders.
Procedure for collection of CSF :
• The person is asked to lie on
their back for the lumbar
puncture.

• Needle is inserted at the L3-L4

intervertebral space and the
CSF is collected in sterile
containers.
CSF is collected in three sterile containers one each for :
• Culture
• Cell count
• Biochemical examination

• Transport of CSF sample should occur at room

temperature for bacterial culture and for virus
isolation the sample must be transported on ice to
preserve viral infectivity.

CSF Examination
Routine Tests (non-microbiological tests)
• The appearance of CSF, total cell counts and protein
and glucose concentration are noted.
Microscopic examination of stained CSF Sample

1. A heaped drop of sediment of a centrifuged sample of CSF is placed on a dry,

clean slide without spreading. This enhances the chances of finding small numbers
of microorganisms. The sediment is stained by Gram stain and is examined
microscopically to detect the presence of microorganisms. Acridine orange may
also be used, brightly fluorescing organisms can be visualised with this dye.

a. Image of a Gram’s stain slide showing pink coloured Gram negative diplococci bacteria
b. Image of a acridine orange stained slide showing bacteria fluorescing orange red against a dark background
Both stains showcase Neisseria meningitidis (causative agent of Meningococcal meningitis)
2. Ziel – Neelsen stained smear is
used to demonstrate tubercle
bacilli.

3. India Ink preparation of CSF is

used to demonstrate capsulated
budding yeast cells of Cryptococcus
neoformans (causative agent of
Cryptococcal meningitis)
4. A wet mount of freshly
collected CSF may demonstrate
motile free-living amoebae.
Brain tissue from an autopsy
sample may also reveal free
living amoebae

Naegleria fowleri in human

brain tissue from a patient
suffering from primary amoebic
meningoencephalitis
Culture
Bacterial Culture :
• Chocolate and blood agar are used. Specimen can also be inoculated in enriched
media like BHI broth or automated blood culture bottle. If there is growth of
bacteria in culture, an antibiotic sensitivity test is performed. Mycobacterium
tuberculosis is cultured in an automated system such as BacT/Alert.

Bacterial growth of Streptococcus pneunoniae(A) and Haemophilus influenzae (B)

on blood agar and chocolate agar respectively.
Fungal Culture :
• This is done on Sabouraud dextrose agar. It is inoculated and incubated at
25°C and organism is identified based on colony/growth morphology.
Birdseed agar is used for culture of Cryptococcus species.

Birdseed agar for cryptococcus species

Culture of free living amoebae :
• A non-nutrient agar medium is used for this purpose.
• Naegleria fowleri can be cultured on non-nutrient agar plates coated
with Escherichia coli. The amoebae will feed on the bacteria, multiply,
and differentiate into cysts within a few days.

PCR- based molecular tests

• Nucleic acid detection in CSF : For suspected microbial infections
including those caused by Mycobacterium tuberculosis and viruses,
PCR-based tests are performed to detect respective nucleic acid DNA
or RNA from CSF. This has now become one of the most confirmatory
diagnostic tool for meningitis.
CLINICAL CASE STUDY
A five year old child was brought to the emergency department with history
of seizures following high grade fever with 1-2 episodes of projectile
vomiting. On examination, he had altered sensorium, and neck rigidity with a
positive Kernig's sign. With the given case study what would be the
appropriate diagnostic tool :

(A) CSF Analysis

(B) Urine Analysis
(C) Stool Analysis
(D) Sputum Analysis
Synovial Fluid
• Synovial fluid is the viscous fluid present in synovial joints.
• It helps in reducing friction between joints.
• Collection of synovial fluid :
It occurs by a procedure known as
Joint Aspiration (Arthrocentesis).

• In Arthritis, joint aspirates are stained to demonstrate bacteria like staphylococcus

aureus and polymorphs.
• They are cultured on blood agar to yield the organism. Blood culture may yield the
organism in around 30% of cases in septic arthritis.
Gastric Aspirate
• Gastric aspirate is the gastric contents collected to mainly try to confirm the
diagnosis of TB by microscopy and culture.

Gastric aspirate can be collected by nasogastric tube

PLEURAL FLUID ANALYSIS
• Pleural fluid is the fluid present in between the parietal
pleura and visceral pleura.
• Normal range- 10-20ml
Indications to collect pleural fluid
• Pleural fluid is collected in cases of pleural effusion.
• A wide range of things can cause a pleural effusion. Some of the more
common ones are:
• Congestive heart failure, when the heart doesn't pump blood to
the body properly.
• In case of liver and kidney disease when fluid builds up in your body
and leaks into the pleural space.
• Lung carcinoma
• Autoimmune conditions- SLE or Rheumatoid arthritis
• Infections. Some organisms that lead to pleural effusion are-
• BACTERIAL CAUSES
• Streptococcal pneumoniae
• Streptococcal pyogenes
• Mycobacterium tuberculosis
• VIRAL CAUSES
• Influenza virus
• Parainfluenza virus
• Respiratory syncytial virus
• FUNGAL CAUSES
• Aspergillus fumigatus
• Amoebic liver abscess
SYMPTOMS OF PLEURAL EFFUSION
• Symptoms of pleural effusion include:
• Shortness of breath
• Chest pain- when breathing in deeply. This is called pleurisy or pleuritic
pain.
• Fever
• Cough
• Confirm pleural effusion by Chest X –ray, CT scan, ultrasound etc
How to collect •

Pleural Fluid
• The pleural fluid should be collected using needle in sterile screw
capped container.
• A minimum of 75ml of fluid needs to be collected before
sending for microscopic examination and culture .
• It should be immediately plated upon receipt .
• Physical appearance -
• Bloody- TB, malignancy
• Black- Aspergillosis , amoebic liver abscess
• Cytology- To detect malignant cells
• Cell count- RBC and WBC count
• Adenosine deaminase test for tuberculosis detection.
• Microscopic examination using gram stain should be done.
• It should be plated upon- Blood agar, Chocolate
agar, MacConky agar etc
• To detect viruses - Antigen , Antibody detection , Molecular method
Transudate and Exudate Fluid
PERICARDIAL FLUID ANALYSIS
• Pericardial fluid is the fluid present in between the parietal and
visceral pericardium .
• Pericarditis is the inflammation of the pericardium and can be caused
due to-
• Streptococcus pyogenes
• Mycobacterium tuberculosis
PERICARDIOCENTESIS
Pericardiocentesis is the removal of pericardial fluid from the pericardial sac with a
needle and syringe.
An intravenous (IV) line may be started and the person may be given medications
prior to the sample collection.
The individual being tested lies down and a healthcare practitioner applies local
anesthetic to the chest.
The practitioner then inserts a needle into the space between the ribs (fifth to sixth
intercostal space) on the left side of the chest and into the pericardial sac and
removes a fluid sample.
An ultrasound may be used to help guide the needle.
• Pericardial fluid is collected via pericardiocentesis or open surgical drainage.
• The specimen stability is as follows:
• Cells may degenerate during storage. Therefore, the pericardial fluid sample for cytopathology
study should be sent to the laboratory as soon as possible in a fresh state or refrigerated at 2-8º
C.
• The volume must be a minimum of 2 mL for each laboratory test.
• Gram stain—for direct observation of bacteria or
fungi under a microscope. There should be no
organisms present in pericardial fluid.
• Bacterial culture and susceptibility testing—
ordered to detect any microorganisms, which will
grow in the culture, and to guide antimicrobial
therapy.
PERITONEAL FLUID ANALYSIS
• Peritoneal fluid is the fluid produced by mesothelial cells in
peritoneum
• Normal volume of this fluid is – 40-50ml
• It is usually straw colored
Ascites
• An abnormally increased volume of peritoneal fluid is called ascites
• This can happen in the following conditions-
• Liver cirrhosis
• Heart failure
• Alcoholic hepatitis
• Pancreatic cancer
• Infections- mycobacterium tuberculosis and klebsiella are few
common infectious causes .
Processing of the sample

• The sample is collected in a sterile

container.
• It is immediately
transported for cytology and
microbiological investigations.
• About 30-50ml of fluid is collected
•THANK YOU