Final Exam: Clin Path B

Four Quadrant Streaking Method

-Sterilize a wire loop by heating it until red hot in a flame, allow it to cool for several seconds. Test for coolness by touching the agar at the edge of the plate. -Pickup a loop full of liquid innoculum or bacteria growth from the surface of an agar plate and starting about one inch in from the edge of the plate, streak lightly back and forth with the loop flat, making close, parallel streaks back to the edge of the plate. -Sterilize the loop and cool again, then with the edge of the loop, lightly make another set of nearly parallel streaks about 1/8 inch apart, in one direction only, from the inoculated area to one side of the uninnoculated area, so that about ½ the plate is now covered. -Flame and cool the loop again and make another set of streaks in one direction, perpendicular to and crossing the send set of streaks but avoiding the first set.

What are the 4 experimental steps of Koch's postulates?

1. The agent of an infectious disease must be present in every case of the disease. 2. The agent must be isolated from the host and grown in vitro (pure culture). 3. The disease must be reproduced when a pure culture of the agent is inoculated into a healthy susceptible host. 4. The same agent must be recovered once again (re-isolated) from the experimentally infected host.

Routine cultures are incubated for ________ hours before initial examination?

18

Most clinical microbiology incubators keep specimens at what temperature?

37 degrees Centigrade (96.6F)

How long should you leave the inoculation loop in the Bacti-Cinerator III to ensure all microbes are destroyed?

5 seconds.

What is the main idea behind Koch's postulates?

A disease is caused by one specific microbe. If the microbe could be isolated, then it might be possible to control the disease.

Pathogen

A disease-causing organism.

What is Koch's Postulates?

A group of four experimental steps which will proved that a particular microbe was the cause of any one disease. Supported the idea that a disease is caused by one specific microbe. If the microbe could be isolated, then it might be possible to control the disease. Isolation of bacteria for the purpose of proving disease was the birth of diagnostic microbiology.

Culture

A liquid or solid medium containing bacteria which have grown (or are growing) in or on that medium.

Cell Wall

A semi-rigid casing which provides structural support and shape to the cell.

Plasma Membrane

A thin sheet of lipid and protein that surrounds the cytoplasm and controls the flow of materials into and out of the cell.

List the different types of media

Agar - a semisolid, gel media Broth - a clear liquid media Plate - a flat, round container of agar Slant - a tube of agar that has been allowed to gel at an angle Nutrient media - grows all types of bacteria Selective media - contains compounds that identify certain characteristics of organisms grown on media Differential media - contains compounds that identify certain characteristics of organisms grown on media

Capsule

An additional outer covering which provides a protective and adhesion function.

Lesions

Anatomically distinguishable changes in body tissues, both gross and microscopic.

Medium

Any solid or liquid specially prepared for bacterial growth.

Disease

Any variation from the normal morphology or physiology of a living organism. Results from various causes (infection, genetic defect, environmental stress etc.)

Rod-shaped bacteria

Bacilli

Anaerobes

Bacteria that only grow in the absence of oxygen (i.e. Clostridium spp.)

What is the major issue with decolorizer (ethanol)?

Be careful not to add too much, this could decolorize all of the specimen, even the gram positive one, giving you a false gram negative result!! However, if you don't use enough decolorizer you could get a false positive result.

Bacterium grow by _______ ________, in which the circular DNA molecule is replicated, then the cell splits into two identical cells, each containing an exact copy of the original cell's DNA.

Binary fission.

Specimen Collection techniques for Anaerobic cultures

Body fluids or aspirates: -Send in a "Cary Blair" gel transport tube. Tissues: -Send in a sterile screw-capped container. -Forward promptly at ambient temperature.

Specimen Collection techniques for Aerobic cultures

Body fluids, aspirates, washes, tissues, sputum: send specimen in a sterile screw-capped containers. -Forward promptly to lab at ambient temperature. Swab specimens: place swab in a culturette transport tube and forward promptly at ambient temperature. Urine: send 10 mL of urine in a sterile screw-capped container or urine culture transport tube with preservative. -Keep specimen refrigerated.

List the sterilizing heat sources:

Bunsen burner, Alcohol lamps, Autoclave, Bacti-Cinerator III.

How do we characterize disease?

By an identifiable group of lesions, clinical signs or symptoms.

Supernatant

Centrifuged fluid.

Examples of Laboratory safety equipment:

Chemical drug vent, Eye wash station, Sharps container, Fire Extinguisher, First-Aid Kit.

Round bacteria

Cocci

Differential Media

Contains additives that cause an observable color change in the medium when a particular chemical reaction occurs. They are useful in differentiating bacteria according to some biochemical characteristic. In other words, the indicate whether or not certain organisms can carry out a specific biochemical reaction during its normal metabolism. Used to distinguish organisms that are closely related.

Enrichment Media

Contains additives that enhance the growth of certain organisms. Useful when the organism you wish to culture is present in relatively small numbers compared to the other organisms growing in the mixture

Appropriate times for each step in Gram staining:

Crystal violet (PURPLE) - 60secs, Lugol's iodine solution - 60secs, Alcohol - decolorizer - 3secs, Safranin (PINK)- 60secs, Water for rinsing.

What are the reagents used in Gram Staining?

Crystal violet (PURPLE) - primary stain, Lugol's iodine solution - mordant, Alcohol - decolorizer, Safranin (PINK)- counterstain, Water for rinsing.

Biohazard Label

Denotes the presence of potentially hazardous biological material → use caution. May be contaminated equipment or biological tissues. NOT radioactive. May be present on bags or container, or designate an entire area.

True or False: Gram-positive bacteria stains red

False: Gram positive stains purple. Gram negative stains pink.

True or False: Oculars are always 10x so the image total magnification is 100 times the objective power.

False: Its 10 times the objective power.

Pili

Fine, hair-like bristles from the cell surface that help in adhesion to other cells and surfaces.

Light microscope

For viewing microscopic objects. Most are binocular design. Most common objectives: 4x, 10x, 40x, 100x. Most use transmitted lighting ("underneath" lighting). Eyepiece lenses (oculars) are always 10x.

What does Lugol's iodine solution do to the membrane?

Forms an insoluble dye-iodine complex .

When placing the specimen on the a slide, the _________ side should be up.

Frosted.

How does alcohol (decolorizer) react with gram positive and negative bacteria?

Gram positive = no effect. Gram negative = Completely removes the outer lipid layer With the outer lipid layer gone, the dye can easily be rinsed out the bacteria due to the thin peptidoglycan layer

What is the primary initial method of differentiating bacteria?

Gram staining.

Selective Media

Has agents added which will inhibit the growth of one group of organisms while permitting the growth of another.

What should you do before the staining process in Gram Staining?

Heat-fix the slide.

The 40x objective is called?

High dry.

Types of Centrifuges:

Horizontal head (swinging bucket/arm), Angled centrifuged head (fixed), Microhematocrit centrifuge.

What is unique about Acid-Fast organism?

In addition to peptidoglycan, the cell wall contains a large amount of glycolipids such as mycolic acid. Characterized by wax-like, nearly impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids.

A solid medium for bacterial growth was developed by ________.

Koch

The 10x objective is called?

Low power.

Refractometer

Measures a fluid's specific gravity or protein concentration by measuring how the fluid refracts or bends light.

Bacteriology

Microbiology of bacteria.

Mycology

Microbiology of fungi.

Virology

Microbiology of viruses.

What is "Streaking out"?

Most important technique to isolate bacteria. Performed on the surface of a solid nutrient medium, the principle being that a single organism physically separated from others on the surface of the medium, will multiply and give rise to a localized colony of descendants.

Acid-fast Staining is important for what 2 Genera?

Mycobacterium and Nocardia.

During smear perparation, Should you add water when using a broth medium?

No.

Disadvantage of the Bacti-Cinerator III

Not intended for use with items such as scalpels, forceps, and test tubes.

Clinical signs

Objectively observable changes in behavior or function.

The 100x objective is called?

Oil immersion.

During slide preparation, if working from a solid medium, add ______ drop of water to your specimen.

One.

Sediment

Particulate matter.

Gram-Negative bacteria stains what color?

Pink

Sometimes an organism may stain both Gram positive and negative, which is called a Gram-variable reaction. A quick test to determine the bacteria's status is:

Potassium hydroxide test.

Quality control (QC) or quality assurance:

Process for assessing the accuracy and reliability of a test system. Usually accomplished by running specimens with known values or outcomes along with patient unknowns.

Gram-Positive bacteria stains what color?

Purple

Aerobic bacteria

Require the presence of oxygen to live

QC programs at a minimum should address:

Requirements for staff proficiency. Equipment maintenance and monitoring. Other standards of operations.

The 4x objective is called?

Scanning

What are the 4 groups of Specialized Media?

Selective Media, Differential Media, Enrichment Media, Combination (selective and differential media).

Bacteria

Single celled organisms classed as prokaryotes which lack defined membrane-bound organelles, including nucleus, mitochondria, Golgi apparatus, and endoplasmic reticulum. Their cellular walls are relatively rigid. They synthesize their own DNA, RNA and proteins, but depend on a host for favorable growth conditions

Who disposes the sharps container once its full?

Special bio-waste handling companies.

Flagella

Specialized appendage attached to the cell that pushes the cell forward and provides motility.

Spiral shaped bacteria

Spirillum

Flexible and undulating bacteria

Spirochete.

Advantage of the Bacti-Cinerator III

Splatters are confined and destroyed with in the heater element.

Types of Microscopes:

Stereoscope/Dissecting microscope, Light microscope, Electron microscope.

What is the difference between the growth patterns of Staphylococcus and Streptococcus?

Streptococcus grows in chains while staphylococcus grows in clusters.

Anatomic pathology

Strives to diagnose disease by concentrating those anatomical (morphological) changes in living tissues (lesions) at the gross and microscopic levels.

Clinical pathology

Strives to diagnose diseases by the use of tests on various body fluids and body waste products (i.e. blood, plasma, urine, CSF, sputum, saliva, peritoneal fluid, thoracic fluid, feces, etc.).

Microbiology

Study of all aspects of the biology of microscopic organisms including structure, growth, reproduction, respiration, and classification.

Symptoms

Subjective changes in behavior or function.

Refraction

The bending of light rays as they pass from one medium to another medium with a different optical density.

A written QC policy should include:

The control materials to be use with each assay. Frequency of use. Required documentation as well as criteria for the acceptability of patient results.

Inoculation

The initial process of adding the cells to the medium.

What is the difference between the cell membrane of Gram positive and Gram negative bacteria?

The lipid bilayer cell membrane of most of the gram-positive bacteria is covered by a porous peptidoglycan layer which does not exclude most antimicrobial agents. Gram-negative bacteria are surrounded by two membranes. The outer membrane functions as an efficient permeability barrier because it contains lipopolysaccharides (LPS) and porins.

Staining

The process of exposing bacteria to colored chemical stains which make them easier to see and aid in identification.

Incubation

The process of maintaining a particular temperature (and/or other desirable conditions) for bacterial growth

Nucleoid region

The site where the large DNA molecule is condensed into a single chromosome packet. There is no surrounding nuclear membrane.

Ribosomes

Tiny particles composed of protein and RNA that are the sites of protein synthesis.

True or False: Images from an Electron microscope are always black and white (grayscale).

True! Images in color are artificially colorized.

How do you calibrate a refractometer for urine?

Use distilled water and set it to 1.000

Sharps container

Used for disposal or sharp materials; primarily syringe needles and occasionally glass slides. Made of strong puncture-proof plastic.

Eye wash station

Used to flush eyes if exposed to harmful materials. Passive water pressure design. Water/saline solution.

Chemical drug vent

Used to mix and draw up chemotherapy.

Stereoscope/Dissecting microscope

Useful in examining objects that can be seen with the naked eye but also requires magnification for detail. Often used to view external parasites like fleas, ticks, and lice (macroscopic). Uses overhead ("reflective") lighting. Objects viewed in 3-D.

Fire Extinguisher

Uses either pressurized dry chemicals, or carbon dioxide.

Colony

Visible mass of microorganisms growing on the agar surface. Different types of microorganisms will frequently produce colonies which differ in their morphological appearance (form, elevation, margin, surface, optical characteristics, and color).

Types of Stains:

Wright stain (Romanowsky, Diff-Quik). Urine Sedi-stain. Gram stain.

When a condition features a unique ____________ of lesions, clinical signs, and/or symptoms, that condition can be given a name.

combination

After streaking, plates are placed ______ ____ in the incubator.

upside down