Specimen collection and

staining
Prepared by: Dr. Ghada Ali

1
 Objectives

1. Describe methods for collecting clinical specimens from different

body sites.
2. Discuss the staining techniques in diagnostic microbiology.
3. Illustrate the different morphological forms of bacteria.

2
Collection Of Microbiological Specimens
 The value and reliability of microbiological reports are directly affected by:
1. The quality of the specimen received by the laboratory.
2. The length of time between its collection and processing.

 The collection of specimens must form part of the department’s SOPs and the
laboratory should issue written instructions to all those responsible for the
collection of specimens from inpatients and outpatients.

3
Collection Of Microbiological Specimens
 Such instructions should include:
1. The amount and type of specimen required.
2. Container to use.
3. Need for any preservative or transport medium.
4. Best time to collect a specimen.
5. Aseptic and safe methods of collection to avoid contamination and accidental
infection.
6. Labelling of the specimen container.
7. Conditions in which specimens need to be kept prior to and during their
transport to the laboratory.
8. Arrangements for processing specimens that are urgent and those collected
outside of normal working hours, e.g. blood cultures collected by medical staff

4
Collection Of Microbiological Specimens
Type of specimen:
 The correct type of specimen to collect will depend on the pathogens to be
isolated, e.g. a cervical not a vaginal swab is required for the most successful
isolation of N. gonorrhoeae from a woman.

 Sputum not saliva is essential for the isolation of respiratory pathogens.

5
Specimen Collection

6
7
Collection Of Microbiological Specimens
Time of collection
 Specimens such as urine and sputum are best collected soon after a patient
wakes when organisms have had the opportunity to multiply over several hours.
 Blood for culture is usually best collected when a patient’s temperature begins to
rise.
 Important: Every effort must be made to collect specimens for microbiological
investigation before antimicrobial treatment is started and to process specimens
as soon after collection as possible.

8
Collection Of Microbiological Specimens
Collection techniques:
 Use a collection technique that will ensure a specimen contains only
those organisms from the site where it was collected.

 If contaminating organisms are introduced into a specimen during its

collection, this may lead to difficulties in interpreting reports.

 A strictly sterile (aseptic) procedure is essential when collecting from

sites that are normally sterile

9
Sterile versus Non-sterile Body Sites
 Sterile body sites:
 These sites normally do not contain any bacteria, so any bacteria found there are
significant
 Blood
 Spinal fluid
 Effusions
 Non-sterile body sites:
 These sites are open to the external environment and normally contain bacteria

 Throat
 Feces

10
Collection Of Microbiological Specimens
 Collect specimens in sterile, easy to open, leak-proof, dry containers,
free from all traces of disinfectant.

 Patients should be instructed in the aseptic collection of specimens and

asked to avoid contaminating the outside of containers.

11
Collection Of Microbiological Specimens
Labelling specimens:
 Each specimen must be clearly labelled with the date and time of
collection, and the patient’s name, number, ward or health center.

 Each specimen must be accompanied by a correctly completed request

form.

12
 Transport of microbiological specimens
 Specimens should reach the laboratory as soon as possible or a suitable
preservative or transport medium must be used.

 Refrigeration at 4–10 °C can help to preserve cells and reduce the multiplication
of commensals in unpreserved specimens.

 Specimens for the isolation of Haemophilus, S. pneumoniae, or Neisseria species,

however, must never be refrigerated because cold kills these pathogens.

13
 Staining techniques in diagnostic microbiology
• Structural details of bacteria cannot be seen under a light microscope due to lack
of contrast and natural pigmentation.
• Hence, it is necessary to use staining methods to produce color contrast and
thereby increase visibility
1. Simple stains: one dye is used (Methylene blue, crystal violet and safranin)
2. Differential stains:
A. Gram stain
B. Acid-fast stain
C. Endospore stain
3. Negative stain

14
Gram stain
 Developed by Hans Christian Gram 1884.
 Pathogenic microorganisms can be classified according to Gram-
staining into two broad groups.
The Gram stain procedure includes:
1. Crystal violet (primary stain)
2. Iodine (Mordant)
3. 95% ethyl alcohol (Decolorizing agent)
4. Safranin (Counterstain)

15
Principle of Gram stain
1. Flood the smear with the basic dye crystal violet for 1 minute and
then rinse with water. Crystal violet, which is called the primary
stain, colors all cells.
2. Flood the smear with an iodine solution for 1 minute and then rinse
with water. Iodine is a mordant, a substance that binds to a dye and
makes it less soluble. After this step, all cells remain purple.
3. Rinse the smear with a solution of ethanol and acetone for 10–15
seconds and then rinse with water. This solution, which acts as a
decolorizing agent.
4. Flood the smear with safranin for 1 minute and then rinse with
water.
16
Principle of Gram stain

17
18
Principle of Gram stain

19
 Gram Gram
positive negative

20
Gram positive cocci

21
Gram positive bacilli

22
Gram negative cocci

23
Gram negative bacilli

24
 The acid-fast stain
▪The acid-fast stain is used to differentiate cells with waxy cell walls as
Mycobacterium tuberculosis appears as Acid fast bacilli

25
 Endospores stain
▪Endospores are stained by the endospore stain procedure.

26
 Negative stains
▪ Indian ink that stain the background and leave the cells colorless are
called negative stains (or capsule stains).

Capsule

27
Negative stains

28
The different morphological forms of
bacteria
Shape
▪Cocci or spherical.
▪Bacilli or rods.
▪Spirochetes are spiral shaped.
▪Vibrio.
▪Pleomorphic, some bacteria are variable in shape.

29
30
 Arrangement
▪Cocci occur in pairs (diplococci) as Pneumococci.

▪Some in chains (Streptococci).

▪Other in grape like clusters (Staphylococci).

▪These arrangements are determined by the orientation & degree of
attachment of bacteria at the time of cell division.

31
32
References
 Bauman, Robert W., Elizabeth machunis-masuoka, and cecily D. Cosby.
Microbiology: with diseases by body system. 4th ed. Benjamin cummings, 2012
Chapter P 96

33
34