MMSC270 Chap 2 Objectives

The Five I’s of Microbiology

1. Inoculation

2. Incubation

3. Isolation

4. Inspection

5. Identification

Inoculation

Introducing a sample into culture media

Incubation

Placing culture under conditions that allow growth

Isolation

Separating one species from others to get pure colonies

Inspection

Observing colonies for characteristics (Size, shape, color)I

Identification

Determining the exact microbe (using tests like stains, biochemical tests, DNA analysis)

What are the three physical states of culture media

• Liquid (broth).

• Solid (agar plates, slants).

• Semisolid (soft agar used for motility tests).

Selective vs. Differential Media

• Selective media: Allows only certain microbes to grow; inhibits others.

  • Example: MacConkey agar (inhibits Gram-positives, allows Gram-negatives).

• Differential media: Shows visible differences between microbes.

  • Example: Blood agar (some bacteria cause hemolysis, others don’t).

Best method for colony isolation

Streak plate method – spreading a sample across an agar plate in streaks to dilute bacteria, so single colonies can grow.

Method:
- loop or sterile tool is dipped into a mixed microbial sample
- microbes are spread across the surface of a solid agar plate in a pattern that progressively thins out the bacteria
- by the end of the streaking, individual cells are deposited far apart enough to grow into separate colonies

Types:
- quadrant streak: plate divided into 4 sections
- T-streak: plate divided into 3 sections

Contributions of 3 pioneers of microscopy

Galileo: developed compound telescope design - one of the earliest microscopes; famous for astronomy and laid the groundwork for optical magnification

Hooke: credited with the term "cell" after observing cork tissue under a compound microscope; publishes Micrographia, which had illustrations of microscopic organisms; improved the compound microscope

Leeuwenhoek: designed a powerful single-lens microscope and was the first to observe and describe bacteria, protozoa, red blood cells, sperm cells, and various other microorganisms; Father of microbiology; refuted spontaneous generation

Resolution:

Ability to see two objects as separate (sharpness/clarity).

Contrast:

Difference in light/dark that makes details visible.

Real image:

Formed by the objective lens, can be projected.

Virtual image:

Seen through the eyepiece (ocular lens), not projected.

Measurement for bacteria and viruses

• Bacteria: Micrometers (µm) → usually 1–10 µm.

• Viruses: Nanometers (nm) → usually 20–300 nm.

Parts of the bright field microscope

1. Ocular Lens (Eyepiece)

Magnifies the real image formed by the objective lens, usually 10x.

2. Body Tube (Head)

Maintains proper distance between ocular and objective lenses for focusing.

3. Nosepiece (Revolving Turret)

Holds the objective lenses and allows easy rotation to change magnification.

4. Objective Lenses

Primary lenses that magnify the specimen.

Usually 4x (scanning), 10x (low power), 40x (high dry), 100x (oil immersion).

5. Arm

Supports the upper parts of the microscope; used for carrying the microscope.

6. Stage

Platform where the slide is placed. Often has stage clips or a mechanical stage to hold and move the slide.

7. Stage Adjustment Knobs

Move the slide left/right and forward/back for precise positioning.

8. Coarse Focus Knob

Moves the stage rapidly up/down for approximate focusing (used with low power objectives).

7. Fine Focus Knob

Moves the stage slowly for precise focusing (used with high-power objectives).

9. Condenser Lens

Focuses light onto the specimen to improve illumination and resolution.

10. Iris Diaphragm

Adjusts amount of light entering the condenser to control contrast.

11. Light Source / Illuminator

Provides light that passes through the specimen; can be a mirror reflecting external light or an internal bulb.

12. Base

Function: Supports the microscope; houses the illuminator.

Purpose of the oil immersion lens

Oil immersion (100x) reduces light scattering, increases resolution, and allows you to see tiny details in bacteria.

Three elements of good microscopy

• Magnification – enlarging the specimen.

• Resolution – clarity of details.

• Contrast – making the specimen stand out.

Visible Light Microscopes (3 types)

• Bright-field: Light passes through specimen.

  • Advantage: Simple, cheap.

  • Disadvantage: Low contrast without staining.

• Dark-field: Special condenser reflects light off specimen at an angle.

  • Advantage: Good for live/unstained microbes.

  • Disadvantage: Low detail inside cells.

• Phase-contrast: Converts light differences into contrast.

  • Advantage: Great for internal structures of live cells.

  • Disadvantage: More expensive.

Bright-field

- most widely used

- forms image when light is transmitted through specimen

- used for both live, unstained material and preserved stained material

Advantages:

- fairly cheap

- easy to learn

Disadvantages:

- limits to 1000x magnification,

- thick specimens lose resolution

- hard to see unstained objects with clarity

Dark-field

- blocks light from entering object lens besides peripheral light

- used to see living cells

Advantages:

- can look at objects while they are still alive

- easy to see

Disadvantages:

- needs special attachment

Phase contrast

Advantages:

- best to view living organisms

- can see internal structure of larger cells

Disadvantages:

- special condenser needed

UV Microscopes (2 types)

• Fluorescence microscope: Uses UV light and fluorescent dyes.

  • Advantage: Specific labeling of structures.

  • Disadvantage: Requires stains/dyes.

• Confocal microscope: Uses lasers + computer to create 3D images.

  • Advantage: High resolution, 3D view.

  • Disadvantage: Expensive, complex.

Fluorescence

- specimen must be stained with fluorescent dye

- UV radiation gives off light that will form own image, usually red, blue, or green

Advantages:

- easy to find small or few objects in a field

Disadvantages:

- need adapter or special uv light source

Confocal

- scans various depths, sharp image focused on one single plane
- captures a highly focused view an any level from the surface to the middle of a cell
- used on fluorescently stained specimens, and live unstained cells and tissue

Advantages:
- sharp images

Disadvantages:
- slow imaging speed

Electron Microscopes: Transmission (TEM)

- detailed structure of cells and viruses
- transmits electron through specimen
- must be sectioned in extremely thin slices and stained or coated with metals

Advantages:
- 200,000x magnification for brightfield
- sees viruses that are very small

Disadvantages:
- expensive: $200,000 for one
- extensive specimen processing

Electron Microscopes: Scanning (SEM)

- used to visualize surfaces of un-sectioned objects
- creates extremely detailed 3D view

Advantages:
- larger sample handling
- surface and 3D images
- large field of view

Disadvantages
- low magnification
- resolution limitations
- sample preparation

Simple staining

the use of a single stain to color a bacterial cell

ex: methylene blue

Differential staining

using specific stains to distinguish different types of cell

ex: gram stain, acid-fast stain

Special staining

use color to highlight and identify specific parts of a bacteria

ex: capsule stain, spore stain, flagella stain

Gram stain

1) cells stained with crystal violet
2) cells stained with an iodine solution
3) safranin applied as a counterstain
4) gram positive bacteria show as purple; gram negative bacteria show as pink

Gram positive vs Gram negative bacteria

Gram-positive:

- thick peptidoglycan cell wall containing teichoic acid

- crystal violet trapped in cell wall shows up as purple

- simpler cell walls

Gram-negative:

- two dimensional peptidoglycan layer and no teichoic acid

- crystal violet washes out with decolorizer

- bacteria show up pink

- more complex cell wall

Acid fast stain

used for identifying bacteria that have a waxy cell wall and are not decolorized by acid-alcohol

Detect Mycobacterium (TB).

Spore stain

malachite green used to detect spores of Bacillus and Clostridium

specific for the endospore coat of various bacteria, typically a firmicute species.

Capsule stain

used to visualize polysaccharide capsules surrounding bacteria (capsule stains india ink)Negative stain

negative stain - capsules may be colorless against a stained background

Flagella stain

used to visualize flagella

- a mordant (binding agent) is used to encrust flagella with stain to a visible thickness.

Fluorescence stain

used in fluorescence microscopy to view things under UV light

Highlight specific microbes/parts with glowing dyes.

used for:
- direct detection of pathogens
- identifying specific bacteria/antigens
- studying cell structure
- rapid diagnosis

Given a Gram stain, describe the morphology and arrangement of the organism in correct format

Color:

- Gram-positive: purple/blue

- Gram-negative: pink/red

Cell morphology (shape):

- Cocci: spherical

- Bacilli (rods): cylindrical

- Coccobacilli: short, oval rods

- Vibrios: curved rods (comma-shaped)

- Spirilla/spirochetes: spiral/helical

Arrangement (pattern of grouping):

Cocci:

- singles

- pairs

- chains

- clusters

- tetrads (groups of 4), - sarcinae (groups of 8 in cubes)

- Bacilli:

- singles

- pairs

- chains

- palisades (side-by-side)

- rosettes/V-shapes

ex: Gram-positive cocci in pairs and chains