MICROBIOLOGY LAB EXAM 1

What Are The Basic Lab Safety Rules?

• wear a lab coat

• wear close toed shoes

• no food or drinks

• tie back long hair

• no cell phones

• before and after each lab, wash your hands and clean bench

Where Do You Dispose Of Biological Solid Waste?

• biohazardous bag on autoclave cart

  • tape up used agar plates

agar plates, swabs

Where Do You Dispose Of Culture Tubes?

• in tube racks on autoclave cart

  • reusable tubes will be autoclaved and recycled

• always carry tubed media by grasping the tube, not the cap

Where Do You Dispose Of Microscope Slides And Needles?

• biohazard sharps container

Where Do You Dispose Of Chemical Waste?

• brown waste bottle

Where To Dispose Of Broken Glass?

• uncontaminated broken glass = in biohazard sharps container

• contaminated broken glass = in red plastic biohazard sharps container

Why Do We Use Biosafety Level (BSL) 1 and 2 Bacteria In The Laboratory?

• they are safe to use if proper safety precautions are used

Who Is Ignaz Semmelweis?

• hungarian physician in the 1850’s who was an early advocate of handwashing in the medical setting to reduced child bed fever

What Is The Purpose Of A Closed And Open Agar Plate?

• closed plate = will test to determine if the media is sterile

• open plate = will be left open for 15 minutes and will test for possible air contamination

What Are The Three Types Of Microbes Commonly Found On Skin?

• diphtheroids = gram positive bacteria

  • corynebacterium

  • propionibacterium acnes

• staphylococci

  • non-pathogenic, coagulase negative (plasma does not clot) staphylococcus epidermidis

  • potentially pathogenic coagulase positive (plasma clots) staphylococcus aureus

• yeast and fungi = break down/degrade lipid secretions from the secretory glands

How Do You Label Media?

• tubes = label on side

• plates = label on bottom

  • initials

  • specimen

  • date

  • temperature

What Is Aseptic Technique?

• work near a flame

• heat flame inoculation tools (loop, needles)

• pass tube opening through flame

• wash hands

• clean bench

set of practices used to prevent harmful microorganisms like bacteria and viruses from contaminating sterile areas or spreading infection

What Is The Colonial Morphology?

• whole colony shape and size = circular, round, irregular, punctiform (tiny), spreading, swarming, embedded

• colony edge (margin) = smooth (entire), wavy, lobed, branched

• colony elevation (topography) = convex, flat, umbonate (raised in center), umbilicate (sunken in center), raised with spreading edge, flat with raised edge, penny-edged

• color and optical properties

  • pigment color = white, yellow, red, gray, colorless

  • optical properties = opaque vs. translucent, dull vs. shiny

• consistency (texture) = mucoid, dry, buttery

S-M-E-C-T

What Are The Steps Of Using The Microscope Properly?

• move microscope from cupboard (if applicable)

• plug in and turn on lamp

• move 4X or 10X into position

• place slide on stage, center over opening stage

• adjust position of oculars to match eye distance

• focus using course adjustment knob

  • bring object into sharpest focus using fine adjustment knob (for 40X and 100X)

• scan specimen to locate an area to examine

• work through objectives, adjusting fine focus and increasing light for each

  • use iris diaphragm when adjusting the amount of light

  • for best resolution make sure condenser is even with the stage

• to use oil immersion lens, rotate nosepiece to halfway between the high dry and oil immersion lenses, place drop of oil on specimen, rotate oil lens (100X) so that the tip is submerged in oil, adjust fine focus and increase light intensity

• when finished

  • rotate oil immersion (100X) objective so that it’s not in contact with slide, remove slide

  • dispose of freshly prepared slides in sharps container

    • permanent slides are returned to storage after cleaning with lens cleaner and drying with paper towel

  • clean oil off lenses (always start by cleaning 4X, then 10X, then 40X then 100X)

    • be sure to use only lens paper for cleaning any optical surfaces

    • to clean oil immersion lens, gently blot optical surfaces with dry soft double folded lens paper, then wet lens paper with lens cleaner and last with dry lens paper again

  • make sure scope is turned off, cord is wrapped properly, stage is down and 4X objective is pointing down

What Are Useful Concepts To Know When Using The Microscope?

• parfocal = characteristics of the microscope that allows you to go from one objective to the other with minimal focus

• total magnification = magnification of the objective lens X magnification of ocular lens

  • example: 40X objective X 10 ocular (eyepiece) = 400X total magnification

• resolution = clarity of an image, the ability of the microscope lenses to distinguish between two points

• refraction = the light bending ability of a medium

Why Is A Streak Plate Used?

• to isolate a colony

What Is A Colony?

• group of cells visible on the agar plate that arise from a single cell

What Are The Most Common Bacterial Shapes?

• rod

• cocci

  • diplo = pairs

  • tetrad = fours

  • sarcina = eights

  • strepto = chains

  • staphylo = clustered

• spiral

D-T-S-S-S

What Is Fungi?

• molds = multicellular fungi made up of intertwining filaments called hyphae

  • hyphae = individual fungal filaments

  • mycelium = mass/network of hyphae

  • septa (or crosswalls) = walls that divide some hyphae into individual compartments, containing organized nuclei and organelles

  • nonseptate/coenocytic = hyphae that lack septa (crosswalls) and are essentially one continuous compartment containing multiple nuclei

• yeasts = unicellular fungi that typically lack true hyphae

  • they reproduce by

    • budding → a new cell grows from the parent cell

    • fission → one cell divides into two

  • pseudohyphae = some yeasts form chains of elongated buds

• dimorphic fungi = live in yeast or mold depending on environment

mold + yeast

What Are The Asexual And Sexual Fungal Spores?

• asexual spores = reproduced by mitotic division and involve one parental cell, they do not require fusion of two nuclei

  • sporangiospores = spores produced inside a sac-like structure called a sporangium

    • can be motile or nonmotile

  • conidia = non-motile spores produced externally on specialized hyphae called canidiophores

    • phialospores

    • blastospores

    • arthrospores

• sexual spores = produced after the fusion of two parental nuclei, followed by meiotic division

  • zygospores = formed by the fusion of nuclear material from two compatible gametangia

  • ascospores = haploid spores formed inside an ascus, which is typically sac-shaped/elongated

  • basidiospores = haploid spores produced externally on aclub-shaped structure called a basidium

  
  

What Is The Purpose Of Heat Fixing?

• it helps the bacterial cells adhere to the glass, kills them, and makes them more easily stained because their proteins become coagulated (clotted)

What Are The Charges Of Basic And Acidic In Simple Staining?

• basic stains (most commonly used) = positive charge

  • crystal violet

• acidic stains = negative charge

What Are The Steps Of The Gram Stain?

• make a smear

  • turn off bunsen burner

• cover smear with crystal violet

  • let sit 1 minute

• rinse with dih2o

• cover slide with iodine for 1 minute (mordant)

  • forms CV:I complex

• rinse with dih2o

• hold slide at 45 degrees

  • slowly add decolorizer until runoff is clear (no more than 10 seconds)

  • for gram positive = dehydrate cell wall, trap CV:I

  • for gram negative = dissolve outer membrane, wash out CV:I

• rinse slide with dih2o

• cover slide with saffranin, 1 minute

• rinse slide with dih2o

• blot dry with bibulous paper

• view: 10x → 40x → oil → 100x

  • record

What Is The KOH String Test For Gram Staining?

• gram positive = will be watery

• gram negative = will be sticky

a quick test used to help distinguish gram-negative bacteria from gram-positive bacteria

What Are Endospores?

• forms when bacteria are stressed

  • dormant form of bacteria

• exosporium = thick protein coat surrounding endospore

• endospore-forming genera

  • bacillus

  • clostridioides

  • clostridium

What Is An Acid Fast Stain?

• has red rods when stained

• has a waxy cell wall containing mycolic acids

• acid fast bacteria genera

  • mycobacterium

  • nocardia

• acid fast bacteria includes these diseases

  • tuberculosis

  • leprosy

to identify bacteria that have a waxy cell wall containing mycolic acid

What Is A Capsule?

• it has a smooth glycocalyx (sticky layer around cell)

• helps bacteria adhere and evade phagocytosis

What Are The Flagella Arrangements?

• monotrichous = one

• lophotrichous = several in one spot

• amphitrichous = one at each end

• peritrichous = several all around