Lecture-08

Sample collection, sample

processing, sample
transport, detection of microbial
populations.
Gram Staining

Prepared by-
Md. Abu Hasnat Abdullah
Lecturer
Department of Public Health
Daffodil International University
 Outline

At the end of the lecture, students will be able to-

▪ Perform microbiological sample collection from the specimens.

▪ Describe the principles of sample storage and transportation.

▪ Describe different techniques for microbial identification and detection such as

gram staining.
 Microbiology sample collection, processing and transportation

There are four steps involved in obtaining a good quality sample for testing:

(1) preparation of the patient,

(2) collection of the specimen,
(3) processing the specimen, and
(4) storing and/or transporting the specimen to the laboratory.
Microbiology sample collection, processing and transportation…
 Specimen Collection
▪ Devices
➢ Use appropriate collection device or specimen container
➢ Sterile swabs- absorbent material on the tip

▪ Collection and transport system:

➢ Sterile, self-contained
➢ Transport medium
➢ Aerobic or anaerobic
 Transport Medium
▪ Transport medium:
➢ Transport medium is a safe and an appropriate way of carrying the clinical
specimens from distances (long or short) for transporting to the lab for
examination.
o It allows organisms to survive.
o It does not allow organism to proliferate.

▪ Example:
➢ For bacteria- Cary Blair
➢ For viruses- virus transport media (VTM)

*[Note. Cary-Blair Transport Medium is a simple, semi-solid, non-nutritive medium used for the
collection and preservation of microbiological specimens. The minimal nutrients in the medium
facilitate the survival of organisms without multiplication]
 Collection of Human Specimens for Diagnostic Purposes
▪ Parameters to be considered when collecting biological specimen:

▪ WHAT: choice of biological specimen depending on clinical stage.

▪ WHEN: optimal moment of collection, maximum time to reach the
laboratory.
▪ HOW: collection procedure (avoid contamination of specimen); instruments
(sterile) etc.
▪ HOW MUCH: adequate quantity for required tests.
 Collection of Human Specimens for Diagnostic Purposes
▪ Minimal information request form:

▪ About patient: Id, age, gender.

▪ About disease: date of onset, suspected diagnosis/ causative pathogen.
▪ About specimen: type of specimen, collection site, time of collection, time
of shipment, storage conditions (if applicable).
▪ About referring physician: contact data (name, phone no).

File: Covid-19 Sample Collection Form

 Collection of Human Specimens for Diagnostic Purposes
Respiratory Tract Specimens

▪ General requirements:

▪ For the patients: In the morning, before feeding, before brushing teeth;
alternatively: at least 4 hours since last meal and teeth brushing.

▪ For medical staff: wear gloves, face protection (mask, eye protection/face
shield), protective lab coat.
 Collection of Human Specimens for Diagnostic Purposes
Respiratory Tract Specimens

▪ Collection of pharyngeal exudate:

▪ Use Dacron or Rayon swab
▪ Tongue blade and good light
▪ Insert swab behind uvula without
touching it
▪ Swab tonsils, posterior pharynx +lesions
9if any)
▪ Avoid touching tongue, cheeks, teeth.
▪ Place swab in sterile tube
▪ Label the swab transport tube with a
unique patient identifier
▪ Transport to lab (RT/2-8ᵒ C)
*RT=Room Temperature
 Collection of Human Specimens for Diagnostic Purposes
Respiratory Tract Specimens

▪ Collection of nasal/ nasopharyngeal

exudate:
▪ Tilt head backwards
▪ Insert flexible fine-shafted polyester
swab into nostril and back to
nasopharynx
▪ Leave in a place a few seconds
▪ Withdraw slowly; rotating motion
▪ Place swab in sterile tube
▪ Label the swab transport tube with a
unique patient identifier
▪ Transport to lab
 Collection of Human Specimens for Diagnostic Purposes
Respiratory Tract Specimens

▪ Sputum collection: An early morning specimen is recommended. A volume

of 5 to 10 ml is adequate and there is no advantage in collecting a larger
volume. The sample should contain recently discharged material from the
bronchial tree with minimal saliva content.

▪ Patient is instructed to take a deep breath and cough up sputum

directly into a wide mouth sterile container of 50-100 ml capacity.
▪ Avoid saliva or postnatal discharge
▪ Tightly cap container so no spillage occurs.
▪ Wash and dry outside of container and label the SPECIMEN
CONTAINER with name and date and time of collection
▪ Specimens must be processed by the laboratory within 24 hours.
 Collection of Human Specimens for Diagnostic Purposes
Respiratory Tract Specimens

▪ Sputum collection:
 Collection of Human Specimens for Diagnostic Purposes
Stool Specimen:
The recovery of bacterial pathogens from fecal specimens will help confirm the
diagnosis of bacterial gastroenteritis as manifested by diarrhea and/or dysentery.

1. Obtain a collection kit containing Cary Blair transport media from the
laboratory.
2. Collect feces from patients as soon after onset of illness as possible, and
before the start of treatment.
3. Transfer a sample one (no more than ounce) of the specimen using the
spatula attached to the container lid into the Cary Blair medium supplied in
the kit and mix thoroughly.
4. For liquid stool specimens, no more than 10 ml should be added to the Cary
Blair medium and mixed.
5. Specimens must be processed by the laboratory within 72 hours of collection.
 Collection of Human Specimens for Diagnostic Purposes
Urinary Tract Specimen:
To minimize specimen contamination on collection, use the "clean catch"
technique..

1. Obtain a Urine Culture Collection Kit from the laboratory.

2. Collect clean catch specimen according to directions listed below:
▪ Wash hands with soap and water, rinse and dry.
▪ WASH area around urethra with soap.
▪ RINSE area with warm water.
▪ VOID- Pass the first portion of urine into the toilet and then pass a
portion (1 ounce) of the remaining urine into a sterile container. Pass the
rest of the urine into the toilet, close and label the container with name
and date. Store samples in refrigerator.
3. Specimens should be delivered to the laboratory within 24 hours for
processing.
Collection of Human Specimens for Diagnostic Purposes
 Collection of Human Specimens for Diagnostic Purposes
Blood Collection:
To minimize specimen contamination on collection, use the "clean catch"
technique..

1. Identify the patient and applying a tourniquet in his hand intended to collect
from.
2. Cleaning the site using alcohol swab or antiseptics.
3. Specimens should be delivered to the laboratory within 24 hours for
processing.
4. Use a sterile needle and syringe for blood collection using venipuncture
method.
5. Dispose the syringe and needle.
6. Store blood in the specific tube.
 Collection of Human Specimens for Diagnostic Purposes
Blood Collection:
 Collection of Human Specimens for Diagnostic Purposes
Blood Collection:
 Precautions during sample collection
Samples will be rejected if they are:

▪ Unlabeled - All specimens MUST have a unique patient identifier.

▪ Insufficient in Quantity - No specimen received, no specimen in container, or
insufficient specimen to perform testing.
▪ Improperly Preserved - Specimens must be received in the transport media
as defined by the laboratory.
▪ Damaged - Specimen leaked or broken in transit.
▪ Too Old - Aged specimens are diagnostically unreliable.
 Methods of Microbiological Detection and Identification
The detection and identification of microorganisms is a significant feature of
microbiology research. It can be applied to many areas such as environment,
industry, and medicine.

▪ Identification methods can be divided into two groups –

▪ Phenotypic methods(This method clarify microorganisms according to
their phenotypic characteristics)
▪ Genotypic methods (These are reliant upon the interpretation of genetic
information)
 Methods of Microbiological Detection and Identification
Importance–
▪ Medical diagnostics and clinical significance: Identifying a pathogen
isolated from a patient.
▪ Food and beverage industries: identifying a microbial contaminant
responsible for food spoilage and fermentation.
▪ Research setting: identifying a new isolate which carries out an important
process.
 Phenotype Detection Method
Morphological Methods:
Culturing microorganisms is still one of the most widely applied techniques in
microbial studies. Different microbe species give rise to colonies that may be
quite distinct to others, which makes it possible to preliminarily identify
microorganisms by observing colonial morphology.

Different bacterial species produce colonies of different colors, sizes, shapes

and textures. It is also the conventional method of identifying the bacterium
based on appearance.
Phenotype Detection Method
 Phenotype Detection Method
Gram Staining:
Gram Staining is the common, important, and most used differential staining
techniques in microbiology, which was introduced by Danish Bacteriologist Hans
Christian Gram in 1884. This test differentiates the bacteria into Gram Positive
and Gram-Negative Bacteria, which helps in the classification and differentiations
of microorganisms
 Phenotype Detection Method
Gram Staining:
Principles: Gram Staining depends on the structures of bacterial cell wall and
cytoplasmic membrane. Gram -positive bacteria have thick peptidoglycan
layer on their cell wall, so they can resist decolorization of primary stain by
alcohol or acetone mixture, and is not stained by a counter stain. But Gram-
negative bacteria have a thin peptidoglycan layer, so they cannot prevent
decolorization by alcohol or acetone mixture, and so they are stained with a
counter stain.
 Phenotype Detection Method
Gram Staining:
Reagents used in Gram Staining:
1. Primary stain: Crystal violet
2. Gram’s Iodine (Mordant)
3. Decolorizer: Acetone or Ethanol (70% or 95%)
4. Counterstain: Safranin
5. Bunsen burner/ spirit lamp
6. Inoculating loop
7. Microscope
8. Distilled water
9. Grease free glass slides
 Phenotype Detection Method
Gram Staining:
 Phenotype Detection Method
Gram Staining:
Procedures:
1. Preparation of the smear: At first take a clean and dry glass slide and make an ideal smear
with supplied sample using a sterile platinum loop. Then allowed to dry.
2. Fixation: Fix the smear by passing slowly the slide 3-4 times through over the flame of spirit
lamp.
3. Primary staining: Cover the smear with crystal violet solution and wait for 30-60 seconds.
Then wash with water.
4. Mordanting: Cover the smear with Gram’s iodine solution and wait for 1-2 minute.
5. Decolorizing: Decolorize the smear with alcohol or acetone and then water is repeatedly till
the color comes out from the smear.
6. Counter staining: Cover the smear with safranin or dilute carbolfuchsin and wait for 30-40
seconds. Then wash with water, dry in air and observe under microscope.
Phenotype Detection Method

Fig. Process of Gram Staining

 Phenotype Detection Method
Gram Staining:
Interpretation:
Gram positive: Blue/Purple Color
Gram negative: Red/Pink Color
 Phenotype Detection Method
Gram Staining:
Examples:
Gram-positive Bacteria: Actinomyces, Clostridium, Enterococcus, Lactobacillus,
Listeria, Staphylococcus, Streptococcus, Streptomyces etc.

Gram-negative Bacteria: Escherichia coli (E. coli), Salmonella, Shigella,

Pseudomonas, Helicobacter, Legionella etc.
 Genotypic Detection Method
Whole-Genome Sequencing:
It provides a comprehensive understanding of the entire genome, which not only
determines the likelihood that genetic material comes from an individual or group
but also suggests additional information on genetic relationships, origin or
susceptibility to specific diseases.

The major processes of whole-genome sequencing workflow are alignment,

variant calling, annotation and calculating related metrics.
 Genotypic Detection Method
Real-Time PCR:
Real-time polymerase chain reaction (rt-PCR), also known as quantitative
polymerase chain reaction (qPCR), is used to amplify and simultaneously
monitor, and quantify or semi-quantify targeted DNA molecules, which is a
conventional technology in biology labs to provide insights into gene expression
and phenotypes of microorganisms on the genetic level.
 Genotypic Detection Method
Real-Time PCR:
Thank You