Infectious diseases are clinically evident diseases that can be transmitted between individuals or species. They are caused by pathogenic microorganisms like bacteria, viruses, fungi, protozoa, or parasites. Laboratory diagnosis of infectious diseases involves direct or indirect methods. The direct method detects microorganisms or their components in patient specimens through microscopic examination, culture techniques, or non-culture methods. The indirect method detects antibodies against microorganisms in the patient's serum. Microscopic examination is often the first step and involves making stained or unstained smears to look for microorganisms. Culture techniques aim to isolate and identify microbes using various media, tests, and molecular methods. Non-culture techniques more rapidly detect specific antigens or gene sequences

2. Infectious diseases
They are clinically evident diseases with the potential of
transmission from one person or species to another.
They result from the presence of pathogenic
microorganisms (very small organisms that are
invisible to the naked eye) that are able to cause disease
in human beings.
Pathogenic micro-organisms include bacteria, viruses,
fungi, protozoa, and multicellular parasites.
Practical microbiology sessions focus on the diagnosis of
these infectious agents.

3. Laboratory diagnosis of
infectious diseases
Direct method
Indirect (serologic) method
Detection of antibodies
against the microorganism in
the patient’s serum.
Detection of:
 microorganisms,
 their structural components
 their products
in specimens collected from the patient (e.g.
urine, blood, sputum, CSF……etc).

5. I) DIRECT METHOD
1)Specimen Collection
A ’good quality’ clinical specimen.
2) Microscopic Examination:
usually before further processing of specimens
3) Microbial Detection:
a) Culture technique:
b) Non-culture technique

6. I) DIRECT METHOD
1) Specimen Collection
A ’good quality’ clinical specimen
 Collecting specimens before the start of antibiotics.
 Choosing the appropriate specimen (representing the infection
site).
 Using sterile containers and avoiding contaminating the
specimen.
 Transporting the specimen properly to the lab. as early as
possible.

7. Specimen Collection

8. I) DIRECT METHOD
2) Microscopic Examination:
usually before further processing of specimens
 stained /unstained (wet)preparations
 different types of microscopes
Staphylococci in pus

9. I) DIRECT METHOD
3) Microbial Detection:
a) Culture Technique:
 Isolation of the organism in pure culture
→inoculating the specimen onto appropriate artificial culture media
followed by
 Identification of the isolate by e.g.




:
microscopic examination
biochemical reactions
reaction with specific antibody (serologic identification of the organism)
DNA probes
Which of these approaches is used and in what sequence depends upon the type of specimen and
organism.
 Antibiotic sensitivity
After growing the organism in pure culture.

10. I) DIRECT METHOD
3) Microbial Detection:
b) Non-Culture Technique:
I.
Identification of a specific microbial antigen such as :

a structural component (e.g. cell wall antigen, capsular polysaccharide…etc) or
a product (e.g. an exotoxin)

by reacting with specific antibody
OR:
II.
Identification of a specific gene sequence
(i.e. nucleic acid of the organism)
by the application of different molecular methods (e.g. PCR, DNA probe).

11. I) DIRECT METHOD

yield more rapid results (minutes or hours)
(do not depend on growth and multiplication of the organism)
However, antimicrobial susceptibility cannot be determined
(although the presence of resistance genes can be determined by
molecular methods).

Non-culture techniques are mainly applied if:


a rapid diagnosis is needed
The microorganism cannot be cultured on artificial
media

A slowly growing micro-organism

13. II) INDIRECT (SEROLOGIC)
METHOD
 Serologic diagnosis of infectious diseases involves the use of known
microbial
antigens
to
microorganism
detect antibodies
in the patient’s serum.
current (active) infection
against the
is diagnosed by the
detection of one of the following
 specific IgM antibodies
 rising titre of specific IgG antibodies (4-fold or greater rise)
 a single high titre of IgG antibodies in certain diseases
Skin tests
N.B.
based on cell-mediated hypersensitivity may also
help in the diagnosis of certain diseases.

14. Skin Test

15. Diagnosis of infectious diseases
Diagnosis of infectious diseases
DIRECT METHOD
DIRECT METHOD
Specimen
Specimen
Microscopical ex.
Microscopical ex.
Culture technique
Culture technique
Isolation on culture media
Isolation on culture media
.Identification e.g
.Identification e.g
••microscopical.ex.
microscopical. ex.
••bioch.Reactions
bioch. Reactions
••DNAprobe
DNA probe
••serology
serology
INDIRECT METHOD
INDIRECT METHOD
(SEROLOGICAL)
(SEROLOGICAL)
Detection of antibodies in serum
Detection of antibodies in serum
••IgM
IgM
••risingtitre of IgG
rising titre of IgG
Non-culture technique
Non-culture technique
••detection of specific antigen
detection of specific antigen
(serology)
(serology)
•• detection of specific gene
detection of specific gene
sequence (mol. tech.)
sequence (mol. tech.)
Antibiotic sensitivity
Antibiotic sensitivity

16. Microscopy

17. Light Microscope

18. Stained Preparations
Bacterial morphology
Unstained preparations
Motility

19. Bacterial Morphology
Size
Shape
Special arrangement
Staining affinity
Spore formation
Capsule formation
Motility

20. Bacterial Shape

21. Bacterial arrangement
Chains.
Pairs (diploids).
Clusters (group).
No special arrangement.

22. Bacterial arrangement
Cocci
Pairs
Chains
Irregular Clusters
22

23. Bacterial arrangement
Bacilli
Pairs
chains
No special arrangement
23

24. Spore formation
 Morphological characters of bacterial spores:
* Shape.
* Position.
* Staining.

25. Bacterial spores

26. Bacterial capsule

28. Staining of Bacteria
Bacteria cells are almost colorless and
transparent
A staining technique is often applied to the
cells to color them →
Their shape and size can be easily
determined under the microscope.
28

30. Smear preparation
S
Fixation
30

31. Smearing out of the sample

32. Types of Stains
1- simple stain:
Single basic dye e.g. Methylene blue
All bacteria take the color of the dye
2- Differential stain:
Two dyes
separated by a decolorizing agent
e.g. Gram stain
& Ziehl-Neelsen stain
3- Special stain: e.g. Fontana stain

33. Differential staining
Principles of differential stain
* Application of the main stain.
• Decolourization.
*Application of the counter-stain.
e.g. Gram stain & Ziehl-Neelsen
stain

34. 1. Gram Stain

35. Components
Primary Stain: Methyl violet + Gram’s iodine
Decolourizing agent: 95% ethyl alcohol
Counter stain: dil. Carbol fuchsin

36. Principle
Primary Stain Methyl violet + Gram’s iodine
All Violet
→
Decolourizing agent: 95% ethyl alcohol
( violet )
( Gram + ve)
Not decolourized
decolourized ( colourless )
(Gram –ve )
Counter stain: dil. Carbol fuchsin→ colourless→pink(Gram -ve )

37. Gram Staining Technique

38. Gm+ve cocci & G-ve bacilli

39. 2. Ziehl-Neelsen
Stain

40. Mycobacterium
A 3rd type of cell envelope (high lipids content of cell
wall)
Not readily stainable with ordinary stains
.
A strong stain e.g., concentrated carbol fucsin + heat.
Resist decolorization by strong mineral acids or
acid-alcohol →
Acid-fast.

41. Components
Primary Stain: Conc. Carbol fucshin
Decolourizing agent: 20% H2SO4
OR
3% HCL in alcohol
Counter stain: methylene blue

42. Principle
Primary Stain Conc. Carbol fucshin → All Red
Decolourizing agent: 20% H2SO4
OR 3%
HCL in alcohol
Not decolourized ( red )
( acid-fast)
decolourized ( colourless )
(non acid-fast)
Counter stain: methylene blue → Colourless →blue
(non acid-fast)

43. Ziehl-Neelsen Stain Technique
1
2
4
7
3
5
6

45. Questions

48. 5.Acid fast bacilli stained by Z-N stain appear:
a) Violet
b) Blue
c) Red
d) Colourless
e) brown
6.The steps of Gram’s stain is as follows:
a) methyl violet / ethyl alcohol / dil. carbol fuchsin / iodine
b) methyl violet / ethyl alcohol / iodine / dil. carbol fuchsin
c) dil. carbol fuchsin / methyl violet / ethyl alcohol / iodine
d)dil. carbol fuchsin / iodine / methyl violet / ethyl alcohol
e)methyl violet / iodine /alcohol / dil. carbol fuchsin