Microbiology Lab Midterm

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Harding University Spring 2014

disinfectants(page vi)

kills vegetative microbial cells but not resilient endospores

sterilization(page vii)

the complete destruction of all living material including endospores

how do you clean up spills?(page vii)

cover with paper towels and disinfect for 20 minutes

Transient flora(page 1)

colonize the superficial layers of skin- more amenable to remove by routine washing

Resident flora(page 1)

...

Nosocomial infection(page 1)

acquired while in the hospital

Universal Precaution #1(page 2)

wash hands before and after dealing with patients and change gloves

Universal Precaution #2(page 2)

wear gloves and gowns

Universal Precaution #3(page 2)

Wear masks and eye wear when needed- mask alone not sufficient if splashing is likely

Universal Precaution #4(page 2)

use mouth piece to resuscitate

Universal Precaution #5(page 2)

discard dirty needles and sharps immediately in sharps container- do not bend, clip, or recap.

Universal Precaution #6(page 2)

clean blood or contaminated spills by 1) putting on gloves 2) wiping up with paper towels 3) washing with soap and water 4) disinfect with a 1:10 solution of bleach for 10 minutes

TSA(page 2)

tryptic soy agar

Fastidious(page 2)

complex nutritional requirements

MSA(page 2)

Mannitol salt agar- detects Staph

Sterile

the absence of all living organisms, including spores

Disinfection

the removal of vegetative life

Brightfield Microscope(page 7)

allows visible light rays to pass directly through th eye without being deflected by intervening filters

Resolution(page 9)

The ability to distinguish two very small or closley spaced objects

Numerical Aperature(page 9)

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

Dimorphic(page 11)

A fungi that's a yeast at 37 degrees but has a mold-like appearance at 25 degrees

Deuteromycetes(page 11)

reproduces asexually

Superficial mycoses(page 11)

infects the skin and hair(ie ringworm or athletes foot)

Cutaneous and subcutaneous mycoses(page 11)

infects the nails and deeper layers of skin

Mold(page 11)

multicellular filamentous fungi

thallus(page 11)

a macroscopic mold colony- composed of mycelia

Mycelia(page 11)

a mat of strands that make up a thallus

Hyphae(page 11)

individual filaments of mycelia in a thallus

Vegitative hyphae(page 11)

grows on the surface of the medium

aerial hyphae(page 11-12)

rise from the surface- often gives rise to spores

spores(page 12)

released by aerial hyphae

Reproductive hyphae(page 12)

when hyphae release spores

Septum(page 12)

hyphal strands are often composed of individual cells sperated by cross walls called septums

Septate(page 12)

A hyphae strand with septum separations

Coenocytic or non-septate hyphae(page 12)

Fungi that lack cross walls and are a continuing mass of cytoplasm containing multiple nuclei

What kinds of agars can fungi grow on?(page 12)

Potato dextrose, sabourand, or sabourand dextrose- simple in composition with a low PH of 4.5-5.6 which kill bacteria

What are fungi dyed with?(page 12)

lacto-phenol cotton blue stain

Sarcodina- amebic dysentery(page 14)

tophozoite: discrete nuclear membrane with some granules, pseudopods, found in the large intestine, isolated in diarrhetic stools
cyst: round with a thick wall- mature cyst has a quadrinucleated chromatoid body that is sausage shaped
1-4 nuclei

Mastigophora- African sleeping sickness(page 14)

trophozoite: crescent shaped, large center nucleus, granular cytoplasm, 1 flagella, infects peripheral blood streams, no cyst

Trophozoites(page 14)

the vegetative forms of parasites

Cysts(page 14)

a resting and metabolically inactive parasite

Mastigophora-dysentery(page 15)

trophozoite:
pear-shaped with sucking disc, 2 bilateral nuclei, clear cytoplasm, 4 pairs of flagella, infects the small intestine, found in diarrhetic stool
cyst:
oval, 2-4 nuclei, cyst wall, axostyle, parabasal body, 4 pairs of flagella within cyst, found in stool

Ciliophora- dysentery(page 15)

trophozoite:
oval, kidney shaped nucleus, vacuolated, cilia, infects the large intestine, found in diarrhetic stool
cyst:
round, thick walls, 1 nucleus, cilia within cyst, found in stool

fungi Candida albicans(page 18)

yeast
thrush and vaginitis

fungi Cryptococcus neoformans(page 18)

prominent capsule
meningitis

fungi Pneumocystisi carinii(page 18)

common in the immune compromised
atypical pneumonia

fungi Trichophyton spp. and others(page 18)

ringworm(tinea)

parasite Ascaris lumbricoides(page 18)

roundworm

parasite Cryptosporidium spp.(page 18)

protozoan
cryptosporiosis
diarrhea

parasite Entamoeba histolytica(page 18)

protozoan
amebic dysentery

parasite Enterobius vermicularis(page 18)

roundworm
pinworm(anus)

parasite giardia lamblia(page 18)

protozoa
giardiasis
diarrhea

parasite Necator americanus(page 18)

hookworm
anemia

parasite plasmodium spp.(page 18)

protozoa
malaria

parasite toxoplasma gondii(page 18)

protozoan
toxoplamosis
dangerous for the preggers
caused by cats

Bergey's manual of determinative bacteriology(page 21)

classified over 7,000 bacteria
first published in 1923

Gracilicutes(page 21)

thin skinned
gram negative

Firmicutes(page 21)

thick skinned
gram positive

Tenericutes(page 21)

soft skin
no cell wall

Mendosicutes(page 21)

archaea
primitive prokaryotes with unusual cell walls and metabolisms

Archaea(page 21)

primitive prokaryotes with unusual cell walls and metabolisms

Strains(page 21)

varying bacterial species

Biosafety levels class 1(page 22)

biological agent that is unlikely to cause human disease( bacillus subtilus, lactobacillus sp.)

Biosafety levels class 2(page 22)

can cause human disease and can be a hazard\
unlikely to spread
effective treatment available
(staphylococcus aureus, hepatitis virus)

Biosafety levels class 3(page 22)

Biological agent that can cause severe human disease and present a serious hazard to workers, may spread but there is a treatment available
(mycobacterium tuberculosis, yersinia pestis)

Biosafety levels class 4 (page 22)

Biological agent that causes severe human disease and is a serious hazard to workers
high risk of spreading
no treatment available
(ebola virus, marburg virus)

Simple stains (page 33)

crystal violet- uniform purple stain
methylene blue- uniform blue stain
reveals size, morphology, and arrangement of cells

Differential stains: gram stain (page 33)

gram + cells are purple
gram - cells are red
differentiates between gram + and gram - bacteria, which is typically the first step in identification

Differential stains: acid-fast stain (page 33)

red acid-fast cells and blue non-acid-fast cells
distinguishes the genera mycobacterium and nocardia from other bacteria

differential stains: endospore stain (page 33)

red vegetative cell
green endospore
highlights the presence of endospores produced by species int he genera bacillus and clostridium

Special stains: negative stain for capsules (page 33)

background is dark with unstained cells
reveals capsules

Special stains: flagella stain (page 33)

flagella become visible for counting and identifying location

Acidic dyes (page 35)

repelled by bacteria

Hanging drop method (page 53)

tests for mobility

obligate aerobes (page 63)

requires oxygen

microaerophiles (page 63)

require low oxygen and higher carbon

obligate anaerobes (page 63)

can't live with oxygen

Dr.Semmelweis

suggested doctors were spreading cadaverous particles to other patients and said doctors needed to wash their hands. He was harassed and sent to asylum.

Lister

began to implement aseptic practices proposed by Semmelweis and received Order of Merit.

Bacteria often get trapped under rings and fingernails. What 2 problems might this pose for the microbiologist?

The first problem is that you might contaminate the culture of which you are preparing if these bacteria get from underneath your fingernails or ring and onto the culture. The second problem is that because bacteria get trapped so easily you can accidentally take bacteria from the lab and spread it, which could be a problem if you are dealing with pathogens.

List two advantages of hand washing over gloves. List two advantages of gloves over hand washing.

Handwashing because you won't build immunity to the gel when needed, handwashing won't kill good bacteria. Gloves are faster and no resident or transient bacteria is transferred from doctor to patient or patient to doctor.

Three agents used for hand hygiene

soap
2% chlorhexidine antiseptic soap
alcohol-based hand gels which usually contain 60-95% ethanol or isopropanol

Know some multi-drug resistant pathogens

Methacillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococcus (VRE), Mycobacterium tuberculosis

An average wavelength of visible light

550 nm

numerical aperture of high power objective lens:

0.65

limit of resolution

wavelength of light/ (2 x numerical aperture)= 423 nm

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

A blue filter may be placed over the light source because the short wavelength of blue light provides maximum resolution

Why is immersion oil important on the oil immersion lens?

By reducing the amount of refraction at this point, more of the light passing through the slide will be directed to the very narrow diameter lens of the high-power objective. The more light, the clearer the image.

Total magnification

objective magnification x ocular magnification

Limit of resolution

wavelength of light / 2x numerical aperture

numerical aperture

mathematical constant that describes the relative efficiency of a lens in bending light rays (it is the objective lens not the oculars we are talking about)

Bergey's Manual of Determinative Bacteriology

7000 bacteria species have been taxonomically classified and documented in it

Gracilicutes

gram negative bacteria

Firmicutes

gram positive bacteria

Tenericutes

lack a cell wall

Mendosicutes

Archaea with unusual cell wall

What would a gram positive smear look like that was halted after the decolorization step? Gram negative smear?

Positive would still be purple. Negative would be colorless

List 2 factors that may make gram positive bacteria appear gram negative.

- if you don't leave the crystal violet on long enough
- if you overuse safranin it will appear more red than should
- using too much acetone alcohol to take off some crystal violet and keep some safranin

What factor could make gram negative bacteria appear gram positive?

- if you don't leave the acetone alcohol on long enough to get rid of all the crystal violet

What is a mordant? What reagent acts as the mordant in the gram negative?

something that hold something in place ex-grams iodine

What chemical components of endospores render these unique structures so resistant to heat?

They have a high concentration of calcium and dipicolinic acid

Define positive and negative chemotaxis?

positive- bacteria goes from areas of low concentration to high concentration
negative- bacteria goes from areas of high concentration to low concentration

Proteus sp. are often termed 'swarmers'. What does this term mean?

They go everywhere- extremely motile

What toxic component are produced from oxygen?

ROI
superoxide- O2 . -
hypochlorite- OCl-
peroxide- O2 -2
hydroxide- OH-
singlet oxygen - 1O2

Anaerobic infections are often facilitated by coexistence with facultative anaerobes. What role do facultative anaerobes play in sustaining anaerobic infections.

obligate anaerobe can't thrive with oxygen and faculitative can grow in either oxygen or no oxygen. if you have both the facultative will take up the oxygen so that the oligate anaerobe can grow now

Given a mixed broth culture of a facultative anaerobe and an obligate aerobe, how would you obtain a pure culture of each based on oxygen requirements?

Take the sample from the broth and add it to both an aerobic media culture and an anaerobic media culture. From the aerobic culture, there should be two different growths of bacteria. Take the first and put it on a split aerobic culture and a split anaerobic culture. Then take the second and put it opposite to the first on both the aerobic and anaerobic culture. Only one of the two bacteria (the anaerobic one) should grow on the anaerobic culture, and you now have two pure cultures of bacteria.
know opposite way too= mixed broth with FA and an O ANA → first round- grow without oxygen and second round grow with oxygen

What would happen if an organism used up all the carbohydrate in a fermentation tube?

Organisms use sugar first for energy but when all the sugar is used up they have to start breaking down proteins. This increases the pH and causes the indicator to turn purple again.

nitrate test

sees if nitrate reduces to nitrite but it can be broken down to other things. if it is pink before zinc that is a + result. if colorless you add zinc. if red-pink it is NEG because nitrate was not reduced to nitrate on its own. if colorless still- nitrate can be reduced to something other than nitrite

Klebsiella

number one source of surgical wound infections

ARE affected by hand gels

MRSA, VRE, mycobacterium TB, and various fungi. Lipophilic viruses: herpes, HIV, influenza, respiratory syncytial, vaccinia virus

AREN'T affected by hand gels

bacterial spores, protozoan oocysts, certain non lipophilic viruses

Resolving Power

The ability to distinguish two very small or closely spaced objects.

buds

New cells forming from parents cells.

dimorphic

A fungi that is a yeast at 37 degrees C but assumes a mold-like appearance at 25 degrees C.

Deuteromycetes

Reproduce slowly by asexual means.

Superficial mycoses

Cause infection of the skin and hair (ie. Ringworm or athlete's foot)

Cutaneous mycoses and Subcutaneous mycoses

Involve the hair 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. These are more common among the immunocompromised.

molds

Multicellular filamentous fungi.

thallus

A macroscopic mold colony.

mycelia

A mat of strands that make up the thallus.

hyphae

The individual strands that make up the mycelia.

Vegetative hyphae

Grow on the surface of the growth medium.

aerial hyphae

arise from the surface of the growth medium.

spores

rise off of the aerial hyphae.

reproductive hyphae

aerial hyphae that release spores.

septum

Cross walls separating the the individuals cells in a hyphal strand.

septate

Hyphae with septum.

Coencytic(non-septate) hyphae

Lacks septum and therefore have a continuous mass of cytoplasm containing multiple nuclei.

potato dextrose agar (or Sabourand agar:
or Sabourand Dextrose (Sab-Dex) agar)

Simple in composition but have low PH (4.5-5.6) which inhibit the growth of bacteria.

lacto-phenol cotton blue

Helps visualize them hyphal and reproductive structures.

trophozoites

Metabolically active, vegetative forms of a parasite.

cysts

Resting and metabolically inactive form of a parasite.

Oxidation/reduction (O/R) potential

When oxygen dissolves in a medium, organic compounds become more oxidized and the medium exhibits a positive O/R potential. As oxygen is consumed by microorganisms the O/R potential decreases allowing anaerobes to grow.

Three ways to achieve anaerobic growth

Agar deeps: dissolved oxygen has been boiled out
Brewer's thioglycolate medium: Contains the reducing agent thioglycolate and resazurin specifically used for the cultivation of anaerobes.
Anaerobe jar: the most commonly used. the jar must be supplemented by a gas-generating system, such as a Gas-pak, which produces both hydrogen and carbon dioxide upon the addition of water

O-F Test: Oxidative Fermentative

differentiates the fermentation of glucose. Uses the PH indicator bromthymol blue which turns yellow when positive. If the tube inoculated with oil is green but the other is yellow it is oxidative, but if both are yellow it is fermentative.

Methyl Red and Voges-Proskauer (MR-VP)

Most coliforms growing in a MR-VP medium initially convert some glucose to acids. Positive tests are red, negative are yellow

Coliforms

gram negative, non-spore forming bacilli that ferment lactose with acid and gas production. Found in high concentrations in the intestines and indicative of fecal contamination in water samples.

Cytochrome Oxidase Test

Identifies the presence of cytochrome C. Changes to blue or red when positive.

Nitrate Reduction Test

determines if nitrate is changed to nitrite. Red is positive.

ONPG test

B(beta)-galactosidase is an enzyme which can cleave a B(beta)-galactoside (ie. lactose) into two monosaccharides. this is positive when it changes from clear to yellow.

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