Micro Lab Exam 1

BSL 1

BSL 1

Microorganisms not known to cause disease in healthy adults. Examples: Bacillus subtilis, Serratia marcescens, Staphylococcus saprophyticus

Standard microbiological practices are followed.

• Personal protective equipment (PPE) is not required.

• A sink must be available for hand washing.

BSL 2

Indigenous microorganisms that can lead to diseases of varying severity in healthy adults.

Examples: Staphylococcus aureus, Streptococcus pyogenes, Salmonella

Standard microbiological practices are followed.

• A sink for handwashing and an eyewash must be available. • Laboratory access is restricted.

• PPE must be worn including lab coats and gloves.

Eye protection and face shields are worn when needed. • Work that may generate aerosols or splashes are performed in a biological safety cabinet/hood. • Contaminated waste is autoclaved. • The laboratory has self-closing doors.

BSL 3

– Indigenous or exotic microorganisms that cause serious or potentially lethal disease through respiratory transmission. Examples: Mycobacterium tuberculosis, Bacillus anthracis, Francisella tularensis

Standard microbiological practices are followed. • A hands-free sink and eyewash must be available. • Locking, double door access separates the lab’s work area from other parts of the facility. • PPE must be worn including lab coats, gloves, eye protection, and respirators. • All work with microorganisms must be performed in a biosafety cabinet/hood. • Decontamination of all waste and lab clothing before laundering. • Laboratory must have negative air-flow (air can not escape to surrounding rooms) and exhaust air cannot be recirculated (must be vented to outside of building after decontamination by ultra-filtration). • Medical surveillance of laboratory employees. Immunizations may be required.

BSL 4

Microorganisms that are dangerous and exotic with high risk of aerosol transmitted infections. Rarely are there treatments or vaccines for these microorganisms, and the diseases they cause are frequently fatal. Examples: Ebola virus, Marburg virus

Standard microbiological practices are followed.

• Change clothing before entering lab. •

A hands-free sink and eyewash must be available.

• The laboratory must be in a separate building or in an isolated and restricted zone of the building.

• All work with microorganisms must be performed in a Class III biosafety cabinet or by wearing a full body, positive pressure suit.

• Decontaminate all materials before exiting lab (including clothes). • Shower upon exiting lab. • Change clothes before exiting lab. • Laboratory must have negative air-flow (air can not escape to surrounding rooms) and exhaust air cannot be recirculated (must be vented to outside of building after decontamination by ultra-filtration). • Medical surveillance of laboratory employees. Immunizations may be required.

Disinfectants can destroy vegetative cells and viruses but may not destroy

endospores. (Due to this limited ability, disinfectants can not be used to sterilize surfaces or equipment.)

(Light Microscopes) Brightfield microscopes

simplest form of microscopy where light is either passed through, or reflected off, a specimen. Illumination is not altered by devices that change the properties of light (such as polarizers or filters). Requires the use of stains to visualize cells.

Phase contrast microscopes

converts the differences in optical density (refractive index) of cells into different shades of brightness (contrast). Allows for the visualization of morphology, external structures, and some internal structures. Stains are not required.

Darkfield microscopes

contrast is created by a bright specimen on a dark background. It is ideal for revealing morphology and external structures, but does not provide a great deal of information about internal structure. Stains are not required.

Fluorescent microscopes

uses high intensity illumination to excite fluorescent molecules (fluorophores) in the sample. When a molecule absorbs photons, electrons are excited to a higher energy level. As electrons 'relax' back to the ground-state, vibrational energy is lost and, as a result, the emission spectrum is shifted to longer wavelengths. Fluorescence emanates from the sample (and not the illuminating light). Requires use of fluorophores.

Immersion oil

Oil with the same refractive index as glass.

Parfocal

The ability of a microscope to remain relatively in focus when changing from lower power objective to a higher power objective.

Numerical aperature

A mathematical expression that describes how the condenser lens concentrates and focuses the light rays from the light source.

Resolving power

The ability of a lens system to show two closely spaced objects as distinct and separate.

Working distance

The distance between the bottom of the objective lens and the slide.

The deciding factor in whether a bacterium will retain crystal violet or not is the

amount of peptidoglycan present in the bacterium's cell wall.

Gram positive

thick layer of peptidoglycan (retain crystal violet); (purple/crystal violet color)

Gram negative

have a thin layer of peptidoglycan (do not retain crystal violet).

When aerobic bacteria grow by respiration, they use

oxygen as a terminal electron acceptor, converting it to water.

(they also produce hydrogen peroxide (H2O2) as a by-product of this reaction.)

Hydrogen peroxide is a

highly reactive oxidizing agent that can damage enzymes, nucleic acids, and other essential molecules in the bacterial cell.

To avoid H2O2 damage,

aerobes produce the enzyme catalase, which degrades hydrogen peroxide into harmless oxygen and water

Strict anaerobes and aerotolerant bacteria lack

lack the enzyme catalase

Facultative anaerobes can grow from

respiration and fermentation

Catalase test

Purpose: Differentiates microorganisms based on their ability to produce the catalase enzyme. (Positive Bubbles) (Negative no bubbles)

Catalase breaks

hydrogen peroxide down to water and oxygen

Oxidase test tests for the presence of

cytochrome oxidase

Cytochrome oxidase

an enzyme in the electron transport chain

Oxidase catalyzes the

transfer of electrons from reduced cytochrome C to molecular oxygen, producing oxidized cytochrome C and water.

Cytochrome oxidase occurs in bacteria that

carry out respiration where oxygen is the terminal electron acceptor;

hence, the test differentiates between those bacteria that have cytochrome oxidase and use oxygen as a terminal electron acceptor from those that can use oxygen as a terminal electron acceptor but have other types of terminal oxidases.

Gram negative cells in KOH

will lyse, releasing their DNA and causing the liquid to become very viscous. Often, "strings" of DNA can be seen adhering to the loop as it is raised from the slide.

If an organism can breakdown carbohydrates by oxidation or fermentation what is usually produced?

acid

The starch macromolecule consists of two constituents

amylose

amylopectin

Amylose

a straight chain polymer of 200 to 300 glucose molecules

Amylopectin

a larger branched polymer of glucose.

Starch molecules

are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade thhem into subunits that can then be utilized by the organism.

Bacteria that hydrolyze starch produce

amylases that degrade the starch molecule into molecules of maltose, glucose, and dextrins that can enter the bacterial cell.

Starch hydrolysis is detected by

Gram's iodine to a starch agar plate (a differential medium, see definition above)

Bile esculin agar is a

selective and differential medium which is used to presumptively identify Enterococcus and group D Streptococcus

Bile esculin is based on

an organism ability to hydrolyze esculin

Bile esculin agar contains

oxgall (bile salts) to inhibit the growth of Gram positive organisms other than Enterococcus and group D Streptococcus.

Bile esculin test

When an organism hydrolyzes the esculin to form esculetin and dextrose, the esculetin reacts with the ferric citrate to produce a dark brown or black phenolic iron complex turning the medium dark brown or black.

Esculin hydrolyzed to

esculet and dextrose

Esculetin reacts with

ferric citrate

Esculetin and ferric citrate produce

phenolic iron complex

The Coagulase test is an excellent definitive test for confirming identification of

Staphylococcus aureus, since 97% of the strains of Staphylococcus aureus have proven to be coagulase positive

If the plasma coagulates,

the organism produces coagulase and is considered coagulase positive