Microbio Lab 1

Front 1

average size bacteria

Back 1

0.5 to 10 microns

Front 2

molecule that makes up bacteria cell wall

Back 2

peptidoglycan

Front 3

3 morphological types of shapes of bacteria

Back 3

rods/ bacilli
cocci/ spherical
helical/ spirals/ curved rods

Front 4

rods/ bacilli ends?
motile?

Back 4

rounded, flat, or tapered ends
both

Front 5

cocci groupings?
motility?

Back 5

may occur singly, in chains, a tetrad (packet 4 cells), or in irregular masses.
most are nonmotile because they lack flagellas

Front 6

spiral bacteria forms
and motility

Back 6

slender spirochaetes (twirly lines) - motile, rotate axial filaments moving in cork screwlike motion
spirillum (thicker spirals with couple antennas on end)
or comma-shaped curved rod/ vibrio (same as spirillum, smaller)

Front 7

axial filaments

Back 7

type of flagella that originate from both ends of the cell and wrap around the cell body

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wide distribution of bacteria includes

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normal flora of humans and animals
disease causing parasites on many organisms
soil and water systems
deep within Earth's crust

Front 9

bacteria are defined primarily by what 2 things

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cellular structure and small size

Front 10

what type of cell structure do bacteria have?

Back 10

simple- it lacks a defined nucleus surrounded by a nuclear membrane

Front 11

bacteria genetic material is..

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primarily supercoiled, circular DNA molecules residing in cell cytoplasm

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what cellular organelles do bacteria lack

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mitochondria and chloroplasts

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how do bacteria carry out photosynthesis and respiration

Back 13

through modifications in their cell membrane

Front 14

ribosomes in bacteria..

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are structurally different than higher cells in synthesizing proteins and inhibited by many broad-spectrum antibiotics

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mold consists of?

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mycelium

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mycelium

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vegetative part of one fungus that consists of mass branching hyphae.
it is analogous to bacterial colony except LARGER, and branching hyphae make it appear fuzzy and powdery

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Ignaz Semmelweis (Lying-In Hospital, Vienna) 1846-7 Observations

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the primary result of the high # cases (~20%) childbirth fever (puerperal sepsis) was the lack of sanitary practices.
-medical students and physicians went directly from dissection and autopsy rooms to patients' bedsides and assiting in deliveries WITHOUT WASHING HANDS.
-women assisted by midwives and nurses (not allowed in autopsies, more sanitary) showed a lower child death rate

Front 18

what were the results of Semmelweis's policy whereby physicians and medical students had to disinfect hands in solution of chloride of line (bleach) prior to examining obstetric patients or assisting in deliveries?

Back 18

a significant decrease of puerperal sepsis, down to ~1%

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nosocomal infections

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hospital-acquired, such as those from the failure of medical students and physicians to scrub their hands before examining patients

Front 20

microorganisms that normally inhabit human skin

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protect from invasion by pathogens and, hence, contribute to overall health

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three groups of normal flora that dwell on human skin

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diphtheroids
staphylococci
yeasts and fungi

Front 22

diphtheroids

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G+
similar to Corynebacterium diphtheriae owing to their variable morphology
however, nonpathogenic

Front 23

example diphtheroids

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Propionibacterium acnes- anaerobic, lives in hair follicles breaking down sebum preventing drying out of skin

Front 24

Staphylococci

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nonpathogenic, coagulase-neg
organisms inhibit pathogens from establishing presence on skin by effectively competing for nutrients on skin and producing inhibitory substances

Front 25

example of staphylococci

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Staphylococcus epidermis- nonpathogenic, coagulase-negative
Staphylococcus aureus- on nose, various parts of skin, and on hands

Front 26

yeasts and fungi

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some are normal inhabitants of the skin that degrade lipid secretions from secretory glands.
spores of transient, saprophytic fungi from environment can be deposited on human and grow out as fungal colonies.
normally nonpathogenic, but some can cause opportunistic infections, especially in immunocompromised patients (chemotherapy, immunosuppressive drugs)

Front 27

example of yeasts and fungi

Back 27

dermatophytes- fungi that infect hair, skin, nails causing athletes foot and related infections in humans

Front 28

transient bacteria may exist on the skin bc..

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the skin has become temporarily contaminated

Front 29

what is effective against transient bacteria on the skin, in killing them

Back 29

washing and antiseptic soaps

Front 30

why is it difficult to remove normal flora from the skin by washing

Back 30

these organisms reside in hair follicles and are entrenched in the skin

Front 31

CFU

Back 31

colony forming unit
measure of viable bacterial or fungal members

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on agar

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organisms grown on exposed surface

Front 33

transient bacteria may exist on the skin bc..

Back 33

the skin has become temporarily contaminated

Front 34

what is effective against transient bacteria on the skin, in killing them

Back 34

washing and antiseptic soaps

Front 35

why is it difficult to remove normal flora from the skin by washing

Back 35

these organisms reside in hair follicles and are entrenched in the skin

Front 36

transient bacteria may exist on the skin bc..

Back 36

the skin has become temporarily contaminated

Front 37

CFU

Back 37

colony forming unit
measure of viable bacterial or fungal members

Front 38

what is effective against transient bacteria on the skin, in killing them

Back 38

washing and antiseptic soaps

Front 39

on agar

Back 39

organisms grown on exposed surface

Front 40

why is it difficult to remove normal flora from the skin by washing

Back 40

these organisms reside in hair follicles and are entrenched in the skin

Front 41

CFU

Back 41

colony forming unit
measure of viable bacterial or fungal members

Front 42

on agar

Back 42

organisms grown on exposed surface

Front 43

on pour plate

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organisms grow throughout medium

Front 44

brightfield microscope

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microscope allows light rays to pass directly to the eye without being deflected by an intervening opaque plate in the condenser

Front 45

how do you carry a microscope

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use both hands to hold the instrument.
one around the neck and one under the base

Front 46

why should it not be carried with one hand

Back 46

because it will dangle at your side and there is danger of collision with furniture or other objects

Front 47

what are the advantages of keeping your workstation uncluttered

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a clear work are promotes efficiency and results in fewer accidents

Front 48

what do you do with the electric cord of the microscope

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do not allow it do dangle in a way that risks foot entanglement and possible tumble of the microscope off the tabletop

Front 49

taking care of the lenses before and after lab

Back 49

at the beginning of each lab period, check the lenses to make sure they are clean
at the end, be sure to wipe any immersion oil off the immersion lens if it has been used

Front 50

what parts on the microscope make up the framework

Back 50

arm and base.
to this all other parts are attached

Front 51

stage

Back 51

horizontal platform that supports the microscope slide

Front 52

mechanical stage

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clamping device on the stage
used for holding and moving the slide around on the stage

Front 53

mechanical stage control

Back 53

to the right of and below the stage

Front 54

what are the 3 lens systems of all compound microscopes

Back 54

oculars
objectives
condenser

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ocular

Back 55

aka eyepiece
complex piece, located at top of instrument
consists of 2+ internal lenses usually w/ magnification 10x
sometimes 2 oculars/ binoculars

Front 56

where objects found

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attached to rotatable nosepiece
makes possible to move them into position over slide

Front 57

scanning objective

Back 57

4x
allows for rapid scanning of microscopic field

Front 58

how is the total magnification of a compound microscope determined

Back 58

by multiplying the power of the ocular lens (10x) times the power of the objective lens used

Front 59

condenser

Back 59

under stage, collects and directs light from lamp to slide being studied
does not affect magnifying power

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diaphragm

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within condenser
regulates amt light reaching slide

Front 61

coarse and fine adjustment knobs

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bring objects into focus

Front 62

how dipter adjustments are made

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first focus with right eye
then on the left eye without any knobs by turning knurled diopter adjustment ring

Front 63

magnification limit of most microscopes

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1000x

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resolving power

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ability to completely separate 2 objects in microscopic field

Front 65

limit of resolution
d

Back 65

distance between 2 objects

function of wavelenth of light (w) used to observe and numerical aperture
= w/NA

Front 66

numerical aperture

Back 66

how condenser lens concentrates and focuses light rays from light source
- the greater loss of refracted light, the lower the NA- resulting in loss of resolving power

Front 67

aprrox limit resolution for light microscopes

Back 67

0.2 micrometers
(objects closer than this cannot be seen as 2 distinct objects

Front 68

bacterial cell size ~

Back 68

one micrometer

Front 69

significance blue filter

Back 69

shorter wavelengths of light provide maximum resolution

Front 70

condenser positino

Back 70

highest possible
allows max amt light enter objective lens

Front 71

diaphragm

Back 71

should be stopped down, but not too much
closing- increases contrast
but reduces NA

Front 72

immersion oil

Back 72

has same refractive index as glass
forms continuous lens system that limits loss light

Front 73

in order to increase magnification

Back 73

resolution must also increase

Front 74

parfocality

Back 74

image on microscope remains in focus when changing from low-power objective to higher power lens

Front 75

using oil immersion

Back 75

focus on high dry first
put oil on slide
rotate oil lens
diaphragm open entirely( no limit of resolving power)
condenser at highest point
blue or green filter best (enhance rp)

Front 76

relationship betwen working distance of objective lens and magnification power?

Back 76

inversely proportional

Front 77

Low power
mag?
Focal Length(mm)?
Working dist (mm)?

Back 77

10x
16.0
8.0

Front 78

high dry
mag?
focal l?
work dist?

Back 78

40x
4.0
.73

Front 79

oil immersion
mag?
fl?
work d?

Back 79

100x
1.8
.16

Front 80

why flame neck of tubes

Back 80

sterilize
create warm air convection currents up and away from opening to prevent entrance dust particles upon which contaminating bacteria reside

Front 81

work area disinfection

Back 81

destroy vegetative cells and viruses but may not destroy endospores

Front 82

how do you sterilize loop or needle

Back 82

insert into bunsen burnerflame until redhot

Front 83

why cool loop/needle before pickin up bacteria?

Back 83

ensures viable cells are transferred

Front 84

ensuring organisms on loop are delivered to broth liquid medium

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twist loop several times

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ensure organisms on loop deliver to agar slant

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drw loop up surface of slant from bottom to top

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stab cultures

Back 86

needle inserts in agar medium by stabbing agar

Front 87

sterilization of and storage of loop/needle

Back 87

reflame after use, never set on lab table,

Front 88

petri plate inoculations

Back 88

barely raise cover slip (diagonally) to protect from contamination in air
loop streaked gently over agar surface w/out disturbing

Front 89

why are inoculated plates incubated upside down

Back 89

to prevent moisture from condensing on agar surface and spreading inoculated organisms

Front 90

what is aseptic technique

Back 90

set of methods intended to supress contamination of cultures and yourselves during manipulations

Front 91

where is label on agar plate

Back 91

bottom

Front 92

to retrieve sample from culture tube with inoculating loop, the cap of the tube is where?

Back 92

removed and held with fingers of loop hand

Front 93

simple staining

Back 93

use of a single stain to color a bacterial cell

Front 94

which dyes work well for simple staining

Back 94

methylene blue
basic fuchsin
crystal violet

Front 95

dyes that work on simple staining have what? and what charge?

Back 95

color-bearing ions, chromophores
positively charge, cationic

Front 96

basic dyes?

Back 96

cationic chromophores attract negatively charged bacteria

methylene blue: methylene+ chloride-

Front 97

acidic dyes?

Back 97

anionic chromophores do not stain bacteria bc electrostatic repelling forces that are involved
eosin = sodium+ eosinate-

Front 98

simple stains are helpful in

Back 98

determining basic morphology
and the presence/absence of certain granules

Front 99

simple staining used on what bacteria?
identifies what?

Back 99

Cornebacterium diptheriae-
pathogenic form causes diphtheria
-demonstrate unique characteristics: pleomorphism, metachromatic granules, and palisade arrangement cells

Front 100

simple staining identifies

Back 100

pleomorphism
metachromatic granules
palisade arrangement

Front 101

pleomorphism

Back 101

irregularity of form/ shape

Front 102

C diphtheriae and pleomorphism

Back 102

C.diph normally rod-shaped
but can also appear club-shaped, sperm-like, or needle-shaped
it is "pleomorphic" and "irregular interchangeably"

Front 103

metachromatic granules

Back 103

distinct reddish-purple granules within cells show when organisms stained with methylene blue.
granules- masses of volutin, polymetaphosphate

Front 104

palisade arrangement

Back 104

parallel arrangement of rod-shaped cells
"picket-fence" arrangement

Front 105

steps of simple stain

Back 105

bacterial smear stained with methylene blue(1min)
wash with water
blot with bibulous paper

Front 106

negative stain use

Back 106

morphology study
characterize external structures- capsules
determining cell dimensions

Front 107

charge of neg stain?
chromophore?

Back 107

acidic
negatively charged chromophore repelled by neg charged bacteria

Front 108

slide image when viewing neg stain?

Back 108

cells as transparent objects against negative/indirect stain dark-colored background

Front 109

negative stain dyes

Back 109

india ink
nigrosin

Front 110

neg stain procedure

Back 110

small drop of dye placed at one end of slide
mix bacteria on wire/ toothpick within dye
use another slide edge to spread film of dye to other side of slide

Front 111

capsule in negative stain

Back 111

usually seen as halo surrounding positively stained cell against dark background

Front 112

why are cell dimensions more accurate in negative staining?

Back 112

bc there is no heat fixation possibly shrinking cells and their capsules severly

Front 113

why is negative staining better for observing spirochaetes

Back 113

bc they are very thin cells that do not readily stain with positive stains

Front 114

what is the goal of the neg staining smear

Back 114

to produce a thick and feather-thin end with an ideal in-between

Front 115

what is a direct stain

Back 115

basic dye (+) attracts neg charge of bacteria

Front 116

indirect stain

Back 116

acidic dye (-) repels bacteria (-) and instead stains background

Front 117

capsule

Back 117

glycocalyx
extracellular slime layer surrounds cells

Front 118

capsule role in pathogenic bacteria

Back 118

protective
prevents phagocytic white blood cells from engulfing and destroying cell
also a means to attach to solid surfaces in environment

Front 119

Streptococcus mutans

Back 119

pathogenic
attaches to surface of tooth by capsular material resulting in formation dental plaque = tooth decay

Front 120

capsule composition

Back 120

most polysaccharides
some polypeptides with unique AA(amino acids)

Front 121

why are bacteria not heat fixed before staining

Back 121

it can destroy or shrink capsule and minimize visibility

Front 122

capsule staining procedure

Back 122

negative stain outlines capsule
gentle heat fixing to adhere cells to slide without destorying capusle
then a positive stain with crystal violet (CV)
final result: capsules appear as halos surrounding purple cells on dark background

Front 123

bacteria used for capsule stain

Back 123

Klebsiella pneumoniae

Front 124

microscopy that produces image of unstained cells similar to negative staining

Back 124

phase contrast microscopy

Front 125

Gram Stain order

Back 125

-heat fix cells
1crystal violet- primary stain, stains cells purple
2Gram's Iodine- mordant, complexes with crystal violet forming insoluble complex with gram+ cells embedded in tissue/ fabric
3 acetone alcohol- decolorizer, leaches dye-mordant complex from g- bacteria, not g+
4 safranin- counterstain, stains g- pink, and g+ remain purple

Front 126

by electron microscopy, you can tell that gram-positive cells

Back 126

have thick layer of peptidoglycan making up cell wall

Front 127

cell wall of gram-negative

Back 127

outermembrane covers much thinner layer peptidoglycan

Front 128

what erroneous results do bacteria older than 16-18 hours give?

Back 128

gram positive cultures can convert to gram-variable or gram-negative.
gram negative bacteria never convert to gram positive!!!!!!!!!

Front 129

thin smears

Back 129

allow observation of individual cells and any arrangement in which cells occur.
can affect decolorization

Front 130

what erroneous results can thick smears give in gram stain

Back 130

false gram-positive bc they entrap primary stain unable to remove with acetone alcohol

Front 131

what is the most critical step in gram stain procedure

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decolorization
time critical, must not be over-applied bc can eventually remove dye-mordant complex

Front 132

bacteria gram stain was perfomed on

Back 132

Staph aureus
Ps aeruginosa

Front 133

St. aereus

Back 133

G+
cocci
clumps

Front 134

Ps aeruginosa

Back 134

G-
rod/bacillus
clumps

Front 135

flagella

Back 135

major organelles of motility in bacteria allow cells to move towards nutrients in environment or away from harmful substances (acids)

Front 136

chemotaxis

Back 136

complicated process of move away from harmful substances (acids)

Front 137

structure of flagella

Back 137

rigid helical structure
~10 microns out from cell
very thin less than ~0.2 microns and below resolution of light microscope

Front 138

individual flagellum

Back 138

rigid filament in form of helix connected to hook attached to shaft inserted in series of rings whose # differ for G+ and G-

Front 139

G + flagellum

Back 139

S and M rings
and L and P rings
in order: M S P L hook filament and shafts between all rings make up basal body

Front 140

rotation of flagellum powered by

Back 140

proton motive force (pmf) when proteins assoc with basal body transport protons across cell membrane - charge differential across memb
pmf- induces S and M rings to rotate rotating everything else
other proteins can reverse direction

Front 141

difference between bacterial flagella and eukaryotic flagella

Back 141

bacteria rotation is like propeller on boat engine
and eukaryotic flagella beat like a whip and actually move within

Front 142

methods of observing motility in cells

Back 142

wet mount
hanging drop technique

Front 143

wet mount procedure

Back 143

drop of viable cells on microscope slide covered with glass observed under phase contrast microscope

Front 144

down side of wet mounts

Back 144

can easily dry out by evaporation, does not allow observation for prolongued periods of time

Front 145

hanging drop technique

Back 145

drop of cells place on cover glass, place over slide with concave depression in center with petroleum jelly delays drying

Front 146

possible arrangements flagella

Back 146

polar- one flagellum
lophotrichous- many on 1end
peritrichous- many all around
amphitichious- many on 2 ends

Front 147

Brownian motion

Back 147

movement caused by currents under cover glass
due to polar bombardment or water currents due to pressure
cells jiggle/ shake, "sweep" across field
no vectorial/ directional movement

Front 148

another method to test motility

Back 148

inoculation semisoft agar medium, concentration .4%

Front 149

semisoft agar medium for motility

Back 149

does not inhibit movement
inoculation with needle
if organisms motile- swim away from line of inoculation
nonmotile bacteria remain on line

Front 150

why semisoft agar med preferred sometimes

Back 150

for pathogenic bacteria, potential for infection in making wet mounts reduced

Front 151

best spot to focus on hanging drop

Back 151

edge of drop
most bacteria are drawn there by surface tension

Front 152

best objective on microscope for hanging drop

Back 152

high-dry (or low power- only 2)

Front 153

disadvantage of hanging drop

Back 153

delay in setup can cause development water condensation decreases clarity and motility in organisms

Front 154

bacteria tested for motility

Back 154

P.vulgaris- motile
-cloudiness visible away from line of inoculation
M. luteus- nonmotile

Front 155

swimming

Back 155

typical random 3d walk of an individual bacterium

Front 156

swarming

Back 156

specialized back and forth movement of motile bacteria growing on solid medium

Front 157

consolidation

Back 157

cells stop and undergo cycle of growth and division so colony has distinct zonations
of swarming, resulting daughter cells continue in the next zones

Front 158

bacteria with cell walls with high lipid content

Back 158

Mycobacterium and Nocardia

Front 159

mycolic acid

Back 159

one of the cell wall complex lipids, waxy material
composed of fatty acids and fatty alcohols with hydrocarbon chains up to 80 carbons in length
affects staining properties, prevents bacteria from being stained by many stains

Front 160

acid fast stain identifies

Back 160

Mycobacterium turberculosis
and M. leprae

Front 161

acid fast stain
Ziehl- neelsen method

Back 161

1 carbolfuchsin- primary stain, mixed with phenol in
2 heat- mordant, makes complex more permeable to m acid
3-acid alcohol- decolorizer does not remove trapped stain (acid-fast cells), negatives are easily decolorized
4-methylene blue- counterstain- added to make non acid fast visbile

Front 162

acid fast results

Back 162

positive- red to pink cells
negative- blue

Front 163

why is acid fast only test gram positive bacteria?

Back 163

because the mycobacteria genus are all gram positive bacillus

Front 164

kinyoun acid fast method

Back 164

modification in which concentration of carbolfuchsin and phenol increased for stain to penetrate
NO heat
No destaining with acid alcohol

Front 165

advantage of kinyoun over siehl-neelsen method

Back 165

no toxic fumes are formed with no heat on primary stain and mordant complex
phenol does not vaporize

Front 166

bacteria tested for acid fast and result

Back 166

M. smegmatis- acid fast, G+, rod, soil, external human genitalia, waxy tend to cling to each other
Staph. aureus- non acid fast coccus, normal flora humans