Good Laboratory Practice Regulations

Know COMMON SENSE!

PASS Method

P-Pull
A-Aim
S-Squeeze
S- Sweep

Aseptic technique

prevents unwanted environmental microorganisms from entering a culture.
is a method that prevents the introduction of unwanted
organisms into a culture or an environment. When changing wound dressings, this is used to prevent possible infection. When working with microbial cultures, this is used to prevent introducing additional organisms
into the culture.

Sterile technique

AKA Aseptic technique

Disinfectant

Chemicals applied to kill microorganisms on surfaces
BLEACH

Antiseptic

preparations of chemicals that are meant to be applied to the skin or other living tissue.

Sterilization

any process that eliminates (removes) or kills all forms of microbial life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media

Selective media

Media that permits growth of certain groups of microorganisms while inhibiting the growth of other microorganisms by the addition of an inhibitory substance.

Differential media

Media that distinguishes between species of bacteria by incorporating chemicals to produce specific kind of growth or change in the medium

Enriched media

Blood and other special nutrients may be added to general purpose media to encourage the growth of fastidious microbes. These specially forfited media are called as enriched media. e.g. Blood agar, Chocolate agar.

Enrichment media

Media used to isolate a bacterium by enhancing growth due to added nutrients. This media is designed for the growth of fastidious bacteria.

Simple stain

the smear is stained with a solution of a single dye which stains all cells the same color.

Differential stain

distinguishes two kinds of organisms. An example is the Gram stain technique.

Colony morphology

The shape, color, and general appearance of an individual colony of bacteria on a plate
often used to identify the species present

Gram stain morphology

Method of differentiating bacterial shapes into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls.

hemolysis

The rupturing of red blood cells

peptidoglycan

Polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria (but not forming the cell wall)

Obligate aerobe

Requires oxygen to grow

Facultative anaerobe

Can use oxygen, but also has anaerobic methods of energy production

coliform

A commonly-used bacterial indicator of sanitary quality of foods and water

Escherichia coli

A Gram negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms

Staphylococcus aureus

A facultative anaerobic, Gram-positive coccus and is the most common cause of staph infections

Oxidation

Loss of electrons or an increase in oxidation state

Fermentation

Process of deriving energy from the oxidation of organic compounds

Lactose

A disaccharide sugar that is formed from galactose and glucose

Amylase

An enzyme that breaks starch down into sugar

Deamination

The removal of an amine group from a molecule

Decarboxylation

A chemical reaction which releases carbon dioxide

Catalase

An enzyme that functions to catalyze the decomposition of hydrogen peroxide to water and oxygen

Oxidase

Enzyme that functions to catalyze an oxidation-reduction reaction involving molecular oxygen as the electron acceptor.

urease

Enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia

Alpha Hemolysis

incomplete clearing
Bacterium partially hemolyses RBC’s with a partial clearing zone around bacterial growth; sometimes with greenish pigment caused by reduction of hemoglobin.

Beta Hemolysis

complete clearing
Complete lysis of RBC’s with total clearing zone around bacterial growth.

Gamma Hemolysis

no clearing
No lysis of RBC’s with no clearing zone around the bacterial growth.

Examples of obligate anaerobes

Mycobacterium tuberculosis
Bacillus

Examples of facultative anaerobes

Staphylococcus
E. coli
Listeria

Blood Agar Plate

Enriched with blood to enhance the growth of fastidious bacteria usually pathogenic. Groups of bacteria can be determined by hemolysis of red blood cells by bacterium.

Mannitol Salt Agar

Selects and differentiates among members of genus Staphylococcus.

Mannitol Salt Agar is selective for...
why?

Selective for gram positive staphylococci because staph is salt tolerant and ferments mannitol sugar.

Acidification indicated by...

phenol red pH indicator which turns from red to yellow

What do pathogenic staphylococci form on mannitol salt agar?

small yellow colonies surrounded by yellow zones.

Eosin Methylene Blue Agar
(EMB Plate)

Selects and differentiates members of Enterobacteriaciae. Differentiates genera of coliform group from other genera of Enterobacteriaciae on the basis of lactose fermentation.

EMB Plate inhibits the growth of...

gram positive bacteria

What is the basis of classification of bacteria according to the gram stain?

the cell wall

Gram Positive Bacteria

have a thick layer of peptidoglycan external to the cytoplasmic membrane. more highly cross-linked peptidoglycan structure
contains Lipoteichoic acids (LTA)

Gram Negative Bacteria

have a thin layer of peptidoglycan located between the cytoplasmic membrane and a second membrane called the outer membrane. This region is known as the periplasmic space.
contain Lipopolysaccharides (LPS)

Primary Stain in Gram Stain
how long do you let stand?

Crystal Violet Solution
30-seconds to a minute

Mordant in Gram Stain
how long do you let stand?

Iodine Solution
30-seconds to a minute

Decolorizer in Gram Stain
how long do you let stand?

20% acetone :80% ethyl alcohol
10 seconds

Counter Stain in Gram Stain
how long do you let stand?

Safranin Solution
30-seconds to a minute

Steps to making a heat fixed smear...

Using a sterilized inoculating loop place one drop of water on a clean glass microscope slide
Using a sterilized inoculating loop place small amount of bacteria on the clean glass microscope slide next to the drop of water.
With the inoculating loop emulsify the bacteria in the drop of water placed on the clean glass microscope slide.
Sterilize the inoculating loop after use.
Dry the smear by putting it on the slide warmer or let the smear air dry.
Heat fix the smear by putting it on the slide warmer for one minute. Be careful not to overheat the slide.

Gram positive reaction

purple in color

Gram negative reaction

red in color

Cocci Morphology

balls

Bacillus Morphology

rods

Coccobacillus Morphology

footballs

MacConkey Agar Results pink

Lactose positive
(Lactose fermentation colony)

MacConkey Agar Results transparent, clear, or colorless

Lactose negative
(Non-lactose fermentation colony)

MacConkey Agar

Selective and differentiates members of Enterobacteriaciae.
Media contains bile salts and crystal violet that inhibits growth of gram positive bacteria

Catalase Test

Bubbles = positive
No bubbles = negative

Oxidase Test

Purple = positive
Light pink or absence of color = negative

Oxidase Positive

Mostly, gram-negative diplococci, gram-negative spiral curved rods

Oxidase Negative

Salmonella, Yersinia pestis, E. coli

Susceptibility

when the antibiotic kills the bacteria

Resistant

when the antibiotic does not kill the bacteria

Most common bacteria causing wound infections is...

Staphylococcus aureus.

Eyepiece

where you look through to see the image of your specimen.

Types of eyepieces

monocular & binocular

Body Tube

the long tube that holds the eyepiece and connects it to the objectives.

Nosepiece

the rotating part of the microscope at the bottom of the body tube; it holds the objectives.

Objective Lenses

scanning, low, medium, high, oil immersion) the microscope may have 2, 3 or more objectives attached to the nosepiece; they vary in length (the shortest is the lowest power or magnification; the longest is the highest power or magnification).

Arm

part of the microscope that you carry the microscope with.

How to carry a microscope...

one hand to hold the arm and the other hand to support the base

Coarse Adjustment Knob

large, round knob on the side of the microscope used for focusing the specimen; it may move either the stage or the upper part of the microscope.

Fine Adjustment Knob

small round knob within the larger course adjustment knob or separate small round knob on the side of the microscope used to fine-tune the focus of your specimen after using the coarse adjustment knob.

Stage

large, flat area under the objectives; it has a hole in it (see aperture) that allows light through; the specimen/slide is placed on the stage for viewing.

Stage Clips

shiny, clips on top of the stage which hold the slide in place.

Aperture

the hole in the stage that allows light through for better viewing of the specimen.

Diaphragm

controls the amount of light going through the aperture

Light or Mirror

source of light usually found near the base of the microscope; the light source makes the specimen easier to see

. Base

supports the microscope

Magnification of Eyepiece

10x

Magnification of Scanning lens (red)

4x

Magnification of Low power lens (yellow)

10x

Magnification of High power lens (blue)

40x

Magnification of Oil Immersion lens

100x

Total Magnification

eyepiece magnification (10x) times objective lens

Pure Culture

composed of only one species of microorganism.

Mixed Culture

there are two or more organisms that have distinct characteristics and can be separated easily.

Contaminants

When unwanted organisms are introduced into the culture

Purpose of Quadrant Streaking

to produce isolated colonies of a microorganism on an agar plate.
AKA streaking for isolation

General Purpose Media

Media that provides enough nutrients in which most any microorganism will utilize for growth. This type of media supports the growth of a wide variety of microorganisms.

Nutrient Agar (NA)

general purpose medium used for the isolation of wide variety of bacteria

Why do we stain?

to increase the contrast
Unstained bacteria appear virtually transparent in brightfield illumination

The most common form of microscopy used in clinical microbiology is...

bright field

How long can a urine specimen sit before it is plated? What can be done if it cannot be plated within this time period?

No longer than 2 hours! or bacterial count will not be valid
May be refrigerated up to 24 hours without being plated

What bacteria make up the normal urethral flora?

Females: diphtheroids, lactobacilli, coagulase-negative staphylocci, alpha streptococci and low numbers of Enterobacteriaceae.
Males: diphtheroids, coagulase-negative staphylococci, and Enterococcus species.

What are the most common organisms causing urinary tract infections?

caused by Escherichia coli and other members of the Enterobacteriaceae

Plasmodium vivax

A protozoal parasite and a human pathogen

Giardia lamblia

A flagellated protozoan parasite that colonizes and reproduces in the small intestine

Lysogeny

Integration of the bacteriophage nucleic acid into the host bacterium’s genome

Transduction

Process by which DNA is transferred from one bacterium to another by a virus

Temperate bacteriophage

Exist within the bacterial cell by lysogeny, within a bacterial cell as a circular DNA in that it exists by replicating as if it were a plasmid and does not cause cell death, or can promote cell lysis during growth resulting in host cell death

Cytolysis

Occurs when a cell bursts due to osmotic imbalance that has caused excess water to move into the cell

Kirby Bauer

A test which uses antibiotic-impregnated disks to test whether particular bacteria are susceptible to specific antibiotics

Use dilution test

Stepwise dilution of a substance in a solution

Phenol coefficient

A measure of the bactericidal activity of a chemical compound in relation to phenol

Nosocomial

Infections that are a result of treatment in a hospital or a healthcare service unit

iatrogenic

Adverse effects or complications caused by or resulting from medical treatment of advice

McFarland standards

a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range.

antibiotic

A substance that kills bacteria or slows their growth

antimicrobial

A substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans

MIC

Minimum Inhibitory Concentration- the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation

Disk diffusion test

Means of measuring the effect of an antimicrobial agent against bacterial grown in a culture

MBC

Minimum Bactericidal Concentration- the lowest concentration of antibiotic required to kill the germ

antiviral

A class of medication used specifically for treating viral infections

MMWR

Morbidity & Mortality Weekly Report.a weekly epidemiological digest for the United States published by the Centers for Disease Control and Prevention (CDC). It is the main vehicle for publishing public health information and recommendations that have been received by the CDC from state health departments.

biological vector

a carrier, especially the animal (usually an arthropod) that transfers an infective agent from one host to another.

fomite

any inanimate object or substance capable of carrying infectious organisms, such as germs or parasites, and hence transferring them from one individual to another.

antibodies

a protein produced by the body's immune system when it detects harmful substances, called antigens.

inflammation

part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. The classical signs are pain, heat, redness, swelling, and loss of function.

phagocytosis

the process of engulfing a solid particle

toxin

a poisonous substance produced within living cells or organisms