Microbiology

Staining for light microscopy

Simple stains (basic (+) dyes absorb
Differential stains
light)
Bind to almost all bacteria irrespective of Combinations
of
dyes
and
other
composition.
treatments
Different binding patterns related to cell
envelope composition
Examples
Examples
Methylene blue
Gram Stain
Malachite green
Gram positive
Crystal violet
Gram negative => when with outer
Safranin
membrane
Carbol Fuschin
Staining for electronic microscopy
Heavy metal ions deflect electrons => internal organization
Diseases caused by microbes
Microbes
Amoebic dysentery
Entamoeba histolytica
Sleeping sickness
Trypanosoma brucei gambiense

16S rRNA gene (only prokaryotic cells)

18S rRNA gene => highly conserved
Measuring
measurement)

growth

(Direct Measuring
growth
measurement)

(Indirect

Total cell count by microscopy (Petroff- Metabolic activity (metabolite)

Hausseer
counting
chamber
or
Hemocytometers)
Automated counters by flow cytometry
Turbidity by spectrophotometer
Viable count (spread or pours plate Dry weight
methods)
=>
Measure
living,
reproducing
population
=> Sample need to be diluted
Filtration (membrane filter retain cells)
Most probable number
Importance of purifying viruses
Determine structures (Larger sizes, more stable than cellular components and have
surface proteins)
Follow/ monitor reproduction
Determine concentration
Techniques for isolating viruses:
1. Centrifugation
Differential/ Gradient Centrifugation
2. Precipitation
Followed by centrifugation

3. Denaturation by changing heat, pH

May inactivate the viruses
4. Enzymatic denaturation
Mainly of cell components leaving viruses unharmed
Viral Assay by enumeration or measure infectious units (Plaque Forming
Units)
Enumeration using electron microscope
PFU through serial dilution and cultivation on agar plates or tissue cultures
Hemagglutination assay (Semi quantitative immunological detection)
No. of viruses > RBCs => agglutination
RBC with various dilutions of viruses => determine the highest dilution of virus
causing RBCs to clump together (hemagglutination titer)
Human diseases caused by viruses
Airbone Diseases
Chickenpox (Varicella)
Shingles (Herpes Zoster)
German measles (Rubella)
Influenza (Flu)
Food Borne Diseases
Viral Gastroenteritis
Infectious Hepatitis (Hepatitis virus)
Poliomyelitis (Polio virus)

Direct contact Diseases

AIDS
Cold Sores (Herpes simplex)
Common Cold (Picornaviruses)
Genetal Herpes (Herpes simplex)
Rabies (Rabies virus)
Hepatitis (Herpes virus)
Arthropod Borne Diseases
Yellow fever (Arbovirus)
Toxins
Tetanus toxin (Clostridium tetani)

Others
Warts and Verrucas (Human Papilloma
Virus)
Skin microbiota/ GI microbiota
Collect sample (Skin sample by sterile swab or facial sample)
Extract DNA from sample
Amplify universal 16S rRNA gene
Sequence all 16s rRNA genes and Compare with database
Transfer to selective growth media
Metagenomics (Seqence all DNA directly)
Extract sequence data from microbial communities as they exist in nature
Fluorescent in Situ Hybridization
with fluorescent tag attached to target => permeabilize cells and so the DNA probe
can enter and allow it to find its matching sequence on 16s rRNA
Denaturing Gradient Gel Electrophoresis (DGGE)
Measure the diversity of a population
Greater pattern complexity => Greater population diversity
Automated antibiotic resistance detection (Vitek)
MIC determination
Bacterial identification based on biochemical profiles
Next generation sequencing

Pyrosequencing technology
Ion Torrent technology
Clinical Microbiology Week 7