Laboratory Exam #1 Study Guide

BSL-1 Organisms

Nonpathogenic

Ex. E. coli K-12

BSL-1 Safety Requirements

Standard microbiology practices

BSL-2 Organisms

Moderate-risk pathogens

Ex. Staphylococcus epidermidis

BSL-2 Safety Requirements

Gloves, eye protection, limited access

ubiquitous

microorganisms are found everywhere

ex. air, water, soil, food, human skin, plants, animals

transient microbiota

-picked up from environment

-easily removed

-more likely to include pathogens

-handwashing primarily removes transient microorganisms

resident microbiota

-normally live on human skin

-difficult to remove

-usually benificial

WHO handwashing technique

-palm to palm

-back of hands

-between fingers

-backs of fingers

-thumbs

-fingertips

-wrists

Calculate percent reduction

250 colonies before

25 colonies after

{(before-after)/before] x 100

=(225/250)x100

=90

proper vs. quick handwashing

Proper handwashing 20 sec, covers all surfaces

Quick handwashing only a few seconds

Ocular lens

number 1 on picture

Objective lenses

Objective Magnification

Scanning 4X

Low power 10X

High dry 40X

Oil immersion 100X

number 3 on picture

Stage

number 4 on picture

holds the specimen

Stage clips

holds the specimen in place

Mechanical stage controls

moves the stage left right, back forward

Condenser

gathers light from the illuminator and concentrates it into a focused cone on the specimen

Iris diaphragm

adjustable aperture located beneath the microscope stage, directly below the condenser

Coarse adjustment

lifts stage up and down to bring specimen in focus

Fine adjustment

focuses on the object clearly

Arm

backbone of the microscope

Base

foundation of the microscope

Light source

Provides light to view the specimens

Immersion Oil

Reduces light refraction

Improves resolution

Used ONLY with the 100X objective

Microscope Storage

• Lowest objective (4X) in position

• Stage lowered

• Oil removed

• Cord wrapped correctly

Protists

• Eukaryotic

• Mostly unicellular

• Possess membrane-bound organelles

• Have nuclei

Ex. Amoeba, paramecium, giardia, plasmodium

Trematoda Group

common name: Flukes

ex: Schistosoma

Cestoda group

common name: Tapeworms

ex: Taenia

Nematoda group

Common name: Roundworms

ex. Ascaris

Zygomycota

Produces: Zygospores

Example: Rhizopus (bread mold)

Ascomycota

Produces: Ascospores

Examples: Saccharomyces, Penicillium, Morels

Basidiomycota

Produces: Basidiospores

Examples: Mushrooms, Shelf fungi, Puffballs

Why Use Aseptic Technique

• Prevent contamination

• Maintain pure cultures

• Protect yourself

• Protect others

Sterile Transfer Steps

1. Sterilize loop

2. Cool loop

3. Flame tube

4. Transfer culture

5. Flame tube

6. Sterilize loop again

Streak Plate Purpose

Obtain isolated colonies in third or fourth quadrant

Pure culture

ne bacterial species

Mixed culture

Two or more species intentionally grown together

Contaminated culture

Unwanted microorganism introduced

Turbid

coudiness of a fluid caused by particles

Sediment

particulate matter that settles in the bottom of aquatic environments

Pellicle

thin outer protein layer that supports cell membrane in many single celled eukaryotes

Flocculent

substance that forms or consists of small woolly masses, like flocculent precipitate

Shape

distinct physical form of microorganisms

ex. coccus

Margin

edge or border of bacterial or fungal colony grown on a solid culture

Elevation

side view of a bacterial or fungal colony grown on a solid culture

ie. how much above the sutface the colony rises

Pigmentation

Natural coloration of microorganisms

Texture

physical consistency and surface charcteristics of a bacterial colony

Simple Stain

Determine: Shape, Arrangement, Size

Uses: basic dyes

ex. crystal violet, safranin, etc

SImple stain result

Cells become colored.

Background remains clear

Negative Stain

Observe: True cell size, Capsule, Delicate structures

Uses: Acidic dyes

Negative Stain result

Background becomes dark.

Cells remain unstained.

Never heat fix.

Basic Dye

Positive charge, stains cells

Acidic dye

Negative charge, stains background

Gram stain steps

1. Crystal violet

2. Iodine

3. Alcohol/acetone

4. Safranin

Gram stain results

Organism Color

Gram-positive Purple

Gram-negative Pink

Gram-positive bacteria

• Thick peptidoglycan

• Retain crystal violet-iodine complex

Gram-negative bacteria

• Thin peptidoglycan

• Lose crystal violet during decolorization

• Take up safranin

Common gram stain Errors

Over-decolorization

→ Gram-positive appears pink

Under-decolorization

→ Gram-negative appears purple

Endospore Stain organisms

Common producers:

• Bacillus

• Clostridium

Endospore Stain reagents

Primary stain: Malachite green

Counterstain: Safranin

Endospore Stain results

Endospores: Green

Vegetative cells: Pink

Acid-Fast Stain bacteria

Contain: Mycolic acids

Example: Mycobacterium

Acid-Fast Stain reagents

Primary stain: Carbol fuchsin

Decolorizer: Acid-alcohol

Counterstain: Methylene blue

Acid-Fast Stain results

Organism Color

Acid-fast Red/Pink

Non-acid-fast Blue

Problem: Gram-positive stains pink

Cause: Over-decolorization

Problem: Gram-negative stains purple

Cause: Under-decolorization

Problem: Smear washes off

Cause: Heat fixing omitted

Problem: Negative stain distorted

Cause: Heat fixed

Problem: No isolated colonies

Cause: Loop not sterilized between quadrants

Problem: Endospores pink

Cause: Malachite green not heated properly

Problem: No bacteria visible in negative stain

Cause: Too few cells or smear too thin

why laboratory safety is essential in microbiology

protect the scientist, prevent release of potentially harmful microorganisms