2. Tools in Microbiology

TOOLS OF MICROBIOLOGY

Microscope instrument used to study MO Optical instrument used to observe tiny objects that

cannot be seen by the unaided human eye.

Simple Microscope - contains only one magnifying lens Anton van Leeuwenhoek

Compound Microscope - contains more than one magnifying lens. Compound light microscope. Hans Jansen and son Zacharias.

Photomicrographs - photographs taken through the lens system of compound microscopes.

PARTS OF A COMPOUND MICROSCOPE

Magnifying Parts:

-To enlarge objects of study

- objectives:

- Eyepiece or ocular objective: variable magnification

- Scanner: 5x magnification, used to study larger

organisms

- Low Power Objective (LPO): 10x magnification

- High Power Objective (HPO): 40x magnification

- Oil Immersion Objective: 100x magnification

Illuminating Parts:- Parts that modify light and illuminate object of

study- Abbe condenser: concentrates light- Mirror: reflects light or uses bulbs as main

light source- Iris diaphragm: regulates the amount of

light that hits the object of study Mechanical Parts:

- Supports the magnifying and illuminating parts- Used to focus the lenses

- Draw tube, body tube, revolving nosepiece, dust shield, arm, stage, stage clips, coarse adjustment knob, base, inclination joint

I. TYPES OF MICROSCOPE

A. VISIBLE LIGHT MICROSCOPY1. Bright- Field Microscope

---used to observe morphology of the organisms.

---does not resolve very small specimens (viruses)2. Dark-field Microscope

---”Dark” background, light organisms---used to detect Syphilis (Treponema

pallidum)

VISIBLE LIGHT MICROSCOPY

3. Phase-Contrast Microscope---Observe dense structures---To facilitate detailed

examination of the internal structures of living specimens.

4. Fluorescent Microscope

---Ultraviolet light

---used to show antibodies

B. ELECTRON MICROSCOPE

1. Transmission Electron Microscope

---Highest magnification (10,000- 100,000x)

---Cellular ultra structure and viruses

---2-D image

2. Scanning Electron Microscope

---Surface structure of cells and viruses

---3-D image

---magnification: (1000-10,000x)

Metric System

– used to describe sizes of microorganisms

1. decimeter 10

2. centimeter 100

3. millimeter 1000

4. micrometer 1M – bacteria, protozoa

5. nanometer 1B - viruses

II. STAINING PROCEDURESOBJECTIVES:

1. Kill the organism2. Preserve morphology3. Anchor smear to slide

A. Simple staining- Aqueous or alcohol solution of a single

basic dye- Used to determine size, shape and morphological arrangement- Ex. Methylene blue

Simple Staining Based on shape:

- Cocci: round or spherical bacteria

- Bacilli: rod-shaped or cigar-shaped bacteria

- Spirals: coiled bacteria

- Spirillum: flexible coiled bacteria

- Spirochetes: rigid, coiled bacteria Based on Arrangement of cells:

- Strepto: bacteria in chains

- Staphylo: bacteria in clusters

- Diplo: bacteria in pairs

- Tetra: bacteria in 4s

B. Differential staining – use of 2 or more dyes that may differentiate one type of organism from one another.

1. Gram Stain - used to classify MO- Dr. Hans Christian Gram (1884)

- Procedure: V I A S a. Crystal VViolet (primary stain) b. Gram’s Iodine (Mordant) c. Alcohol (Decolorizer) d. Safranin (Counterstain)

Differential staining

2. Acid-Fast Stain (Ziehl-Nielsen)- Binds strongly to the bacteria that

have a waxy material in their cell wall- Used to identify Mycobacterium, Nocardia

- Procedure: C A M a. Carbolfuchsin (Primary stain) b. Acid-alcohol (Decolorizer) c. Methylene Blue (Counterstain)

Gram staining Gram (+) Gram (-)

Color Blue Pink Pink Red

Peptidoglycan Thick Layer Thin layer

Techoic acid in cell wall

Present Absent

Lipopolysaccharide in cell wall

Absent Present

C.Structural Stains- Observe capsules, spores, flagella

1. Negative stain

- demonstrate the presence of capsules

- capsule (unstained halo) around bacterial cells against dark background.

2. Endospore stain

- Malachite green

3. Flagella stain

- Carbolfuchsin