MICROBIOLOGY-METHODS OVERVIEW OF BASIC MICROBIOLOGY

MICROBIOLOGY METHODS

1. Diagnostic Microbiology
Identification of Microbes

Lecture: 1

2. Out lines
•What is expected out of this course ???
•At the end of this course, you will be able to apply…………
•Conventional/ Molecular diagnostic methods
•Diagnostic Microbiology Techniques
•Some basic principles of microbiology testing
•(Follow a specimen through the lab)

•Task of the methods – to make the microorganisms visible and measurable

3. Component of Diagnostic Microbiology
•the analysis of sample, the synthesis of results (of several samples), and the clinical consultation
•Together these form the basis for
•Diagnosis
•Therapy
•Infection control

The basic principle is
- Clinical diagnosis or assessment
- Collecting and transporting specimens
- Microscopy
- Cultural diagnostic methods
- Some basic principles of microbiology testing
- Culture Sensitivity
- Non-cultural diagnostic methods
- Virological diagnosis
- Immunological methods

4. Major Group of the Microbial World
•Bacteria
•Fungi
•Algae
•Parasite
•Viruses

•Major features
•Small
•Diverse (appearance, genetics)

5. •The method microbiologist use fall into two categories:

Phenotypic (conventional) : morphology (micro and macroscopic)

Genotypic: genetic techniques

6. Phenotypic Methods
‘Old fashioned’ methods via biochemical, serological and morphological are still used to identify many
microorganisms.
•Examining specimens to detect isolate and identify pathogens

•Phenotypic characteristics:
•A- Morphology : Microscopic (wet preparation – Staining preparation)

• Macroscopic (Cultural Characteristics) are traits that can be accessed with the naked
eye e. g appearance of colony including shape, size, color of the colony (pigment), speed of growth and
growth pattern in broth- nutrient requirements for the growth of the organism Growth on different
laboratory conditions and media- Can determine whether the microbe is a bacteria, fungus, or protozo)

B- Biochemical/ Physiology characteristic are traditional for microbes identification
•Biochemical reactions (Metabolic differences) include enzymes (catalase, oxidase,
decarboxylase),fermentation of sugars, capacity to digest or metabolize complex and sensitivity to drugs
can be used in identification- physical factors such as temperature, pH and the incubation period.
C- Immunological tests: serological tests are great value in the diagnosis of many bacterial, fungal and
viral infections.

D- Animal Pathogenicity: to detect toxins and virulence- uncommon techniques used in the identification

•E- Phage Typing: procedure for characterizing and detecting some strains of bacteria by their reaction
(susceptible or resistant), may used in outbreak infections- uncommon techniques used in the
identification process.

•F- fatty acid profiles: routinely used to identification of mostly anaerobic bacteria- non fermentative
gram negative, yeast

7. Phenotypic Methods
Disadvantages
Poor discriminatory power
Difficulties in typing
Not provide enough information about microorganisms for today's needs.

8. A- Morphology
1- Microscopy: Microorganisms can be examined microscopically for
A- Size of individual cells
B- Bacterial motility: Hanging drop method
C-Cells Shape
D- Cells arrangement
E- Useful to be taken into account
F- microscopic structures and characteristics ( such as flagella)
Morphology and staining reactions of bacteria:
1-Simple stain: one dye is used e.g methylene blue stain – reveals shape- size- cell arrangement

The most commonly used stain in diagnostic microbiology is the Gram stain

2- Differential stain: 1- Gram stain: differentiation between Gm+ve and Gm–ve bacteria
. Primary stain (Crystal violet)
. Mordant (Grams Iodine mixture)
. Decolorization (ethyl alcohol)
. Secondary stain ( Saffranin)

Some microbes have unique characteristics that can be detected by special staining procedures

3- Acid fast reaction (Ziehl-Neelsen stain) staining acid fast bacilli

4- ( spore stain): bacterial endospores

5-Structural stains –reveal certain special structures of cell parts not revealed by conventional methods
such as: capsule (capsule stain - flagellar stains- granules (volutin )

This used to give an initial presumptive identification

9. Specimen Preparation for Optical Microscopes
•Wet mounts and hanging drop mounts–allow examination of characteristics of live cells: size,
motility, shape, and arrangement

• Fixed mounts are made by drying and heating a film of specimen. This smear is stained using dyes
to permit visualization of cells or cell parts.

10. Staining
•Dyes create contrast by imparting a color to cells or cell parts
•Basic dyes –cationic, positively charged chromophore
•Acidic dyes –anionic, negatively charged chromophore
•Positive staining –surfaces of microbes are negatively charged and attract basic dyes
•Negative staining–microbe repels dye, the dye stains the background


Staining reactions of dyes

11. Microscope Types
1- Light Microscope (digital)

•2- Stereo microscope

•3- Dark field Microscope

•4- Electron Microscope
•(transmission- Scanning)

1-
2
3
4

12. Microscopic
Morphology
Fungi
Bacteria
Bacteria- dark field
microscope

13. Transmission electron microscope image
of influenza virus
Protozoa
Virus

14. Microbe Stains