Microbiology Lab Midterm Moore

Disinfection

involves the destruction of vegetative microorganisms but not endospores, on inanimate surfaces or objects

the disinfectant in class is a

10% sodium hypochlorite solution (bleach)

Sterilization

complete destruction of all living material including endospores

Autoclave

instrument used to sterilize objects which utilizes high temperature and pressure

Dead week

time in which the lab final will be taken

Asepsis

the state of being free from disease-causing contaminants or, preventing contact with microorganisms

What to do in the event of an accident or spill (in order)

a. Notify fellow students so they may leave immediately

b. Cover spill with paper towel and disinfectant for at least 20 minutes

c. Thoroughly clean area with disinfectant to avoid widespread contamination

d. Place used paper towels in disinfectant reservoir and wash hands thoroughly afterwards

Transient flora

colonize the superficial layers of the skin and are more amenable to removal by routine hand washing

Resident flora

Part of an individual's normal flora, more anchored to deeper layers of the skin and are more resistant to removal

Nosocomial Infection

health-care associated (acquired) infections

opportunistic infections

infections that occur in individuals who do not have healthy immune systems

Universal precautions

CDC issued guidelines to reduce the risk of disease transmission

Fastidious organisms

organisms that have complex nutritional requirements

Tryptic Soy Agar (TSA)

Type: a common, nutrient-rich growth medium used for growing and maintaining bacterial cultures

Purpose: Cultivation of non-fastidious bacteria

Mannitol Salt Agar (MSA)

NaCl - 7.5%. Media is selective for salt tolerant bacteria and inhibits the growth of most microorganisms

Mannitol (a fermented alcohol)- when fermented, the pH drops and the pH indicator in the medium causes colonies to turn yellow.

Compare and contrast Semmeweis and Lister

Semmelweis was a Hungarian physician who observed the spread of disease in 1847. During this time, it was common for physicians to go directly from the autopsy room directly to their OB-GYN duty without washing or scrubbing their hands. This practice led to the development of childbed fever. Semmelweis noticed that infant mortality decreased when doctors left the hospital on vacations or extended leaves. Since nurses were not allowed into the autopsy room, women cared for by nurses had a lower mortality rate. Semmelweis hypothesized that "cadaverous particles" were responsible and implemented a policy requiring physicians to wash their hands with chorine of lime prior to patient contact. He was made fun of for his idea and was sent to an asylum after suffering a nervous breakdown. He died in 1865, the same year Lister implemented aseptic practices. Lister eventually received the Order of Merit award for his work.

Which agent used for hand washing is most efficacious for reducing the number of bacteria on hands?

alcohol based gels

List 3 commonly used agents for handwashing

non-antimicrobial soap, 2% chlorhexidine antiseptic soap, and alcohol-based hand gels (containing 60-95% ethanol or isopropanol)

Describe the proper method of hand washing

Wash hands thoroughly for 30 seconds using soap and warm water. Rinse hands thoroughly and dry using a paper towel. Turn off faucet using hand covered with paper towel. Follow with alcohol base gel and rub in until dry.

List the 6 universal precautions

i. Wash hands before and after patient contact. Wash hands after removal of gloves. Change gloves between patients. Do not keep gloves on for an extended period of time

ii. Wear gloves and gowns if there is a high possibility of soiling hands, skin, or clothing with body fluids.

iii. Wear mask and face shield if body fluids are likely to be splashing.

iv. Use disposable mouthpiece/ airway during CPR

v. Discard used needles and other sharp articles into a puncture proof container. Cannot bend, clip or recap needles

vi. Clean spills of blood or body fluids by:

-Putting on gloves or other protective equipment needed

-Wiping with disposable towels

-Washing with soap and water

-Disinfecting with a 1:10 solution of bleach and water allowing to stand for at least 20 minutes

-Do not make bleach solution more than 24 hours beforehand

Why are the CDC universal precautions important?

keeps transmission of pathogenic bacterial organisms to a minimum which protects hospital personnel and patients

One what body fluids does the universal precautions apply?

blood

List two advantages of hand washing over gloves

removing transient flora and less clumsy

List two advantages of gloves over hand washing

provide barrier against a pathogen, protect patient from normal flora

Brightfield microscope

- microscope that allows visible light rays to pass directly through to the eye without being deflected by intervening filters in the condenser

Ocular lens

remagnifies the image formed by the objective lens

Objective lenses

primary lenses that magnify the specimen

Stage

holds the microscope slide in position

Condenser

focuses the light through the specimen

Diaphragm

- controls the amount of light entering the condenser

Illuminator

light source

Total magnification

product of ocular magnification and objective magnification

resolving power (resolution)

ability to distinguish between two very small or closely spaced objects

Limit of resolution

minimum distance by which two small ofjects can be separated and still be perceived as separate objects

Numerical aperture

is a mathematical constant the describes the relative efficiency of a lens in bending light rays

Parfocal

lenses are adjusted so that the specimen remains almost in focus after the microscopist rotates the nosepiece to utilize a different objective lens

Wet mount

observation of particulate objects or living organisms in an aqueous solution

Buds

new fungal cells formed from parent cells

Pseudohyphae

short chains formed by the detachment of buds

Dimorphic

fungi that may be yeasts at 37°C but have a mold-like appearance at 25°C

Deuteromycetes

reproduce solely by asexual means

Superficial mycoses

cause infections of the skin and hair (ringworm & athletes foot)

Cutaneous mycoses

involve nails and deeper layers of the skin

Subcutaneous mycoses

involve nails and deeper layers of the skin

Systemic mycoses

cause infections of the subcutaneous and deeper tissues such as the nervous system, lungs and genital area

molds

- multicellular filamentous fungi

Thallus

macroscopic mold colony

Mycelia

a mat of strands that compose the thallus

Hyphae

individual filaments within the mycelia

Vegetative hyphae

hyphae that grow on the surface of the growth medium

Aerial hyphae

rise from the surface of the growth medium

Spores

produced by the aerial hyphae

Reproductive hyphae

aerial hyphae that give rise to spores

Septum

cross walls that separate individual cells in hyphal strands

Septae

hyphae that are composed of individual cells separated by septum

Nonseptate hyphae (coenocytic)

- fungi which lack a cross walls and as such are a contiuous mass of cytoplasm containing multiple nuclei

Potato Dextrose agar

medium that is selective for fungal growth that are simple in composition but have a low pH that inhibit the growth of bacteria

Sabourand agar

medium that is selective for fungal growth that are simple in composition but have a low pH that inhibit the growth of bacteria

Sabourand dextrose agar (Sab-Dex)

- medium that is selective for fungal growth that are simple in composition but have a low pH (4.5-6.5) that inhibit the growth of bacteria

Lacto-phenol cotton blue

stain used to help visualize the hyphal and reproductive structures of molds

Guidelines for how to put away the microscope

a) Both hands should be used to transport microscope with one hand securing the base

b) Review the parts of the microscope and their function

c) Turn the fine adjustment knob no more than one turn forward or backward

d) Never touch the ocular lens or objective lens with anything but lens paper

e) Never use immersion oil with any objective but the oil immersion lense (100X)

f) Never remove a slide while the immersion objective lens in in the light path as this may scratch the lens

g) Never decrease the distance between the slide and the 40x or 100x objective while looking through the eyepiece. Start as close to the object as possible and move slowly away until the object comes into focus

h) Never force any part of the microscope

i) If the slide is put upside down on the stage, the specimen cannot be seen using the 100x objective

Before putting away microscope, always:

j) Lower stage or offset the objective to remove the slide

k) Plays low power objective in the light path

l) Remove all traces of oil from 100x

m) Remove oil from other parts of the microscope center the moveable part of the mechanical stage

n) Grip the microscope with both hands when you return it to the cabinet

o) Place the microscope far enough back in the cabinet to prevent contact with the cabinet door

Microscope parts

Light source

Substage condenser

Iris diaphragm

Objective lens

Ocular lens

Ocular lens setting

Mechanical stage

Coarse adjustment knob

Fine adjustment knob

Light source

may be directly connected to the microscope or separate. If separate, the light must be reflected using a mirror underneath the condenser. Blue filter may be placed over the light source because the short wavelength provides maximum resolution

Substage condenser

collects and directs the light through the slide into the objective lens. Should always be kept just beneath the stage and never used for adjusting light intensity. Must be kept close to the stage when visualizing bacteria

Iris diaphragm

controls the amount of light entering the objectives. Higher magnification requires a higher light intensity than lower power. Closing the diaphragm increases image contrast and decreases resolution

Objective lens

magnifies and resolves the specimen. 4 objectives. 4x, 10x, 40x (high-dry), 100x (oil immersion)

ocular lens (eyepiece)

(10x-20x) to magnify the image formed by the objective. These can be moved to adjust for individual interpupillary distance.

Ocular lens setting

turn slowly or physically separate and align with interpupillary distance

Mechanical stage

holds slide in position

Coarse adjustment knob

quick, approximate focusing. used to bring objects into focus

Fine adjustment knob

slow ultimate focusing of the specimen. DO NOT USE FOR GENERAL FOCUSING.

Limit of resolution equation

Limit of resolution=(Wavelength of light (λ))/(2 x numerical aperture)

Total magnification equation

Total magnification= (ocular magnification)x(objective magnification)

An average wavelength of visible light is 550nm and the high power objective lens on many microscopes have a numerical aperture of 0.65. What is the smallest object that can theoretically be seen?

423 nm

How does the inclusion of a blue light filter change resolution?

The short wavelength blue light provides maximum resolution.

What is Brownian movement?

The random movement of microscopic particles suspended in a liquid or gas, caused by collisions with molecules of the surrounding medium. False movement

Why is immersion oil important for on the oil immersion lens?

the small working distance does not allow enough light to enter the lens. the oil directs more light into the lens by limiting the loss of light

List the three categories of eukaryotic microbes

a) Fungi

b) Parasitic Protozoa

c) Parasitic Helminths

Bergey's Manual of Determinative Bacteriology

- manual published in 1923 that taxonomically classifies and documents bacterial species

The 4 major groups recognized by Bergey's Manual

Gracilicutes, Firmicutes, Tenericutes, and Mendosicutes

Gracilicutes

Thin skinned (gram negative)

Firmicutes

Thick skinned (gram positive)

Tenericutes

Soft skin (no cell wall)

Mendosicutes

archaea, primitive prokaryotes with unusual cell walls and metabolism

Genus

part of the scientific name (along with the species name)

Specific epithet (species)

second half of the scientific name

Strains/types/vars.

differences within a bacterial species

biotypes (biovars)

A biotype is a strain of bacteria distinguished from other strains of the same species by its physiological properties

morphotypes (morphovars)

a strain of bacteria distinguished from other strains of the same species by its morphology (shape)

serotypes (serovars)

Serotype bacteria distinguish different strains of the same species by their surface antigen.

Biosafety level (BSL)

categorization of microbes into classes based on their pathogenicity. Used to determine the precautions necessary to protect the public, and safety equipment needed when handling these organisms

Rules for writing names of organisms

Genus and species name is written and underlined or typed and italicized

BSL Class 1

biological agent that is unlikely to cause human disease

BSL Class 2

biological agent that can cause human disease and might be a hazard to workers; it is unlikely to spread to the community; there is usually effective prophylaxis or treatment available

BSL Class 3

biological agent hat can cause severe human disease and presents a serious hazard to workers; it may present a risk of spreading to the community, but there is usually effective prophylaxis or treatment available

BSL Class 4

biological agent that causes severe human disease and is a serious hazard to workers; it may present a high risk of spreaing to the community and there is usually no effective prophylaxis or treatment available

Class 1 example

Bacillus subtilus

Class 2 example

Staphylococcus aureus

Class 3 example

Mycobacterium tuberculosis and Yersinia pestis