Lecture 2 Demonstration of Microorganisms

Question 1

Pathogenic organisms can often be directly visualized by microscopic examination of patient specimen

Answer 1

Sputum
Blood
Urine
Cerebrospinal fluid

Question 2

Gives rapid and inexpensive results without waiting for culture

Answer 2

Microscopy

Question 3

Can provide 1st screening step in arriving at a specific identification

Answer 3

Microscopic morphology and staining characteristics

Question 4

Dye that binds to a cellular structure and gives it color
Unstained bacteria may be difficult to detect with light microscope

Answer 4

Stain

Question 5

Negatively charged, binds to positively charged structures like proteins
E.g. acid fuchsin, eosin, picric acid, nigrosine, India ink

Answer 5

Acidic dyes

Question 6

Positively charged
Since surface of bacterial cells msre negatively charged, _ commonly used in bacteriology
E.g. methylene blue, crystal violet, safranin, malachite green

Answer 6

Basic dyes

Question 7

Classification of stains

Answer 7

Simple stains
Differential stain
Special stain

Question 8

Uses a single dye
Size, shape, arrangement of cells
E.g. methylene blue, crystal violet

Answer 8

Simple stains

Question 9

Uses two or more dyes to distunguish different types or structures of bacteria
Distinguishes gram + and gram -
E.g. mycobacteria, nocardia

Answer 9

Differential stain

Question 10

Identifies specialized structures
Exhibits the presence of flagella, endospores, capsule
E.g. Shaeffer-Fulton method, India ink

Answer 10

Special stain

Question 11

The most common and useful staining procedure
Separates bacteria into 2 classifications according to composition of cell walls
Most, but not all, bacteria are stainable and fall into one of these two groups
E.g. mycoplasma - lacks cell walls; gram-variable bacteria

Answer 11

Gram Stain

Question 12

Applications of Gram Stain

Answer 12

Important therapeutically
- gram + and gram - bacteria have differing susceptibilities to antibiotics
- guide to initial therapy until definitive ID
Sometimes diagnostic
- morphology of stained bacteria
- e.g. spore-forming bacilli in blood of cattle

Question 13

Applications of Gram Stain (2)

Answer 13

Blood smear of anthrax case - Gram + bacilli are seen
Aids interpretation of culture results
- e.g. specimen shows organisms under the microscope but appear sterile on culture media which suggests:
1. Fastidious organisms - bacteria with complex nutrient requirement
2. Fragile organisms - may not survive transport
May provide only clue to nature, variety, and relative number of infecting organisms

Question 14

Limitations of the Gram Stain

Answer 14

High number of microorganisms required
Liquid samples with low numbers of microorganisms (CSF)
- requires centrifugation to concentrate pathogens
- pellet then examined after staining

Question 15

Used to identify organisms that have waxy material (mycolic acids) in cell wall

Answer 15

Acid-fast stain

Question 16

Most clinically important acid-fast bacteria

Answer 16

Mycobacterium tuberculosis

Question 17

Also occurs in animals and may have clinical importance

Answer 17

Nocardia sp.

Question 18

One of the simplest microscopic methods
Background is stained while organisms remain clear
Determines if organism has gelatinous capsule
Able to visualize bacteria as large, transparent capsules that displace II particles
Useful in detecting cryptococcus neoformans in CSF

Answer 18

India ink

Question 19

Stained background
Capsule appears transparent/translucent

Eosin, nigrosin, india ink can be used to stain background
Crystal violet to stain bacterial cells

Answer 19

India ink

Question 20

Technology producing images of very small things with high clarity and magnification

Answer 20

Microscopy

Question 21

Two broad categories of microscopy

Answer 21

Light microscopes
Electron microscopes

Question 22

Magnification should be coupled with

Answer 22

Good resolution

Question 23

Distance at which two objects can be viewed as separate or distinct objects

Answer 23

Resolution

Question 24

Units in Microscopy

Answer 24

Micrometers or microns (mu m) = 10^-6 m
Nanometers (nm) = 10^-9 m
Angstrom (A) = 10^-10 m

Question 25

Involves use of optical lenses and light radiations

Answer 25

Light microscopy

Question 26

Light microscopy can be further categorized as

Answer 26

Bright field m Dark field m Phase contrast m Fluorescence m

Question 27

Invented by Anton van Leeuwenhoek Single lens, bi-convex Not common nowadays, used for dissection purposes

Answer 27

Simple microscopes

Question 28

Multiple lenses Credited to Zaccharias Janssen Uses visible light as source of illumination

Answer 28

Compound microscope

Question 29

Brightly illuminated by focused natural sunlight or white light Results in bright field image of a stained specimen

Answer 29

Bright field m

Question 30

For examining live microorganisms sudpended in liquid Only light reflected by specimen enters objective lens

Answer 30

Dark field m

Question 31

Internal structures of living microorganisms can be examined without fixing or staining Enhanced contrast

Answer 31

Phase contrast m

Question 32

Uses UV light Specimen can be stained using fluorescent dyes Useful for diagnostic and ID purposes

Answer 32

Fluorescence m

Question 33

Phenomenon in which fluorescent substance absorbs short wavelengths of light and emits a light of longer length

Answer 33

Fluorescence

Question 34

Uses electron beam (instead of light) and electromagnets (instead of lens) Provides high resolution

Answer 34

Electron microscopes

Question 35

Two types of electron microscopes

Answer 35

Transmission e m Scanning e m

Question 36

Objects can be seen to order of a few angstrom Ability to study small details in the cell, near atomic levelsuses electromagnetic lenses to focus electrons into a very thin beam 2d, black and white images

Answer 36

Transmission e m

Question 37

Relies on electron interactions at the surface Produces image of the surface Creates 3d images About 50,000x magnification

Answer 37

Scanning e m