Detecting microorganisms techniques

Question 1

What are common techniques used in detection of micro-organisms?

Answer 1

Microscopy
Gram stain
Acid fast stain
Culture
PCR
Serology

Question 2

How does microscopy allow for the detection of micro-organisms?

Answer 2

Enables analysis of size, shape & motility

Question 3

What are the different types of microscopes & what do they involve?

Answer 3

Bright field- uses light & lenses to magnify wet or stained slides- oil emersion sense improves resolution
Dark field- has dark background- useful for observing movements & thin cells e.g. spirochetes
Fluorescence- used to see substances that are naturally fluorescent or have been stained w/ fluorescent dye
Electron- uses beam of e- & magnets to focus the beam- have higher magnification & resolution

Question 4

How is a culture grown?

Answer 4

The micro-organism in a broth, is diluted using serial dilutions
The diluted culture is spread over the agar plate
Left for 24 hours at around 25°C
NOTE- some organisms e.g. viruses, need cell cultures to grow

Question 5

How is the culture used to identify the micro-organism?

Answer 5

Identified through their characteristics & biochemical properties:
Cell shape, size & arrangement i.e. pairs chains.
Ability to grow under aerobic / anaerobic conditions
Growth requirements
Ability to produce enzymes
Ability to metabolise sugars through oxidation or fermentation.
Ability to use a range of substrates for growth.

Question 6

What does PCR test do? Why is it useful?

Answer 6

Amplifies a specific DNA sequence millions of times
Useful for diagnostic work for organism that are hard to culture

Question 7

Explain the steps of performing a PCR?

Answer 7

1. Denaturation- Solution is heated to 95°C. heat breaks the hydrogen bonds in the DNA & separates it into single strands.
2. Annealing- Sample is cooled to 50°C. allows DNA primers to bind to the separate DNA strands.

3.Extension- Occurs at 70-80°C. DNA nucleotides from an added solution pair w/ the separated strands with help from DNA polymerase.

4.Repeat- The DNA forms a new complementary strand of DNA (double stranded). Cycle is then repeated lots of times.

5- Analysis w/ electrophoresis- DNA from sample compared w/ DNA of pathogen.

Question 8

What is serology? When is it useful?

Answer 8

Study of serum & other body fluids
- serum is derived from blood
Useful for pathogens that are hard to culture e.g. treponema palladium (causes syphilis) or viruses

Question 9

What do serological tests do?

Answer 9

Look for antibodies & antigens in patient’s blood
Look for IgM & IgG antibodies
- IgM produced in large quantities after infection
-IgG produced after IgM on subsequent infections

Question 10

Name 3 different serological methods

Answer 10

1. ELISA- enzyme linked immunosorbent assay
2. Agglutination
3. Precipitation