microbio lab

simple microscope

uses only a single lens

light microscopes

use light to view specimens

electron microscope

uses a beam of electrons and magnets to allow higher degrees of magnification

compound microscopes

employ ultimate lenses for better magnification

total magnification

magnification of ocular lens x magnification of the objective lens

pure culture

contains only one type of microorganism

subculturing

microorganisms being transferred from one medium to another

aseptic transfer

requires subculturing be performed with sterile instruments and under sterile conditions to prevent contamination

streak plate technique

reduces the number of organisms on the surface on an agar plate

importance of streak plate technique

allows different species in the culture to be spread far apart enough that the individual cells can multiple without touching

streak plate technique decreases the cell density on a plate,

leading to the isolation and separation of bacteria by dividing the plate into quadrants and streaking the bacteria from one to another

colony morphology

the macroscopic observation of the colony’s growth patterns on an agar plate and includes the size, shape, pigment, form, margin appearance and the elevation of the colony

criteria of colony morphology

size, pigment, optical property, surface, form, margin, and elevation

size

small (pinpoint), medium, large (pinhead)

colony morphology refers to

the form and structure of the colonies on the plate (macroscopic)

cell morphology refers to

the form and structure of the cells when stained (microscopic)

coccus/cocci

spheres

bacillus/bacilli

rod-shaped

vibrios

slightly curved rods

coccobacilli

short rods

spirrillum/spirilla and spiirochetes

spiral-shaped

pleomorphic

multiple shapes

diplo-

cells arrange as pairs

staphylo-

cells arrange as grapelike clusters

strepto-

cells arranged in chains

tetras

a quartet of cells

sarcina

two quartets of cells that form a 3D cube

the process of heat-fixing

dentures cellular proteins, which allows the amino acid side chains to adhere to the slide

agar cultures

on solid media the bacteria will present a colonies on the surface. a drop of water is applied to the center of a slide & the bacteria are mixed with the water

broth cultures

applied directly to the dry slide

direct stain

stains the specimen but not the background. the light passes through the background and colored cells are easily visible

negative stain

stains the background but not the specimen

simple stain

stains a bacterial smear w a single dye

differential stain

uses at least 3 chemical reagents on a smear

differential stain reagents

primary stain, decolonizer, counterstain

primary stain

initially gives color to all cells

decolorizer

gives a color contrast based on the chemical makeup of the cell; will decolonize one type of cell while other cells maintain the primary stain color

counterstain

gives a constraining color from the primary stain to cells that were decolorized

gram stain

most famous differential stain

purpose of gram stain

used to differentiate bacteria based on their cell wall composition

steps of gram stain

1. primary stain: crystal violet

2. mordant: iodine

3. decolorizer: 95% ethanol

4. counterstain: safes in

capsule stain

protective coating around some pathogenic bacteria that protects them from phagocytic activity of the host

selective media

used to select and isolate specific types of bacteria by containing chemical substances that allow the growth of a particular organism while inhibiting the growth of other organisms

example of selective media

Mannitol salt agar (MSA)

differential media

contains chemical compounds that allow bacteria to be distinguished between each other based on their reactions to the compounds on the media ; doesn’t inhibit growth

differential media produces

a characteristic change in the appearance of bacterial growth and/or the media its growing on

example of differential media

blood agar

selective/differential media

contains ingredients that inhibit/select for a certain type of bacteria and components that allow you to differentiate between the bacteria that grow

hemolytic activity

the breakdown of red blood cells by disrupting their cell membranes

gamma hemolytic (nonhemolytic)

organisms produce no lysis of red blood cells

appearance of gamma hemolytic organisms

no change to the medium

alpha hemolysis

partial lysis of red blood cells

appearance of alpha hemolysis

greenish halo around the colony due to the heme iron oxidation of the RBC in the agar

beta hemolysis

the complete lysis of red blood cells

beta hemolysis appearance

clear halo surrounding the colonies

beta hemolysis is the typical pathogenic forms of

staphylococci and streptococci

blood agar

differential medium that serves as an enrichment ingredient for the growth of fastidious bacteria but almost anything will grow on it

blood agar is used

to distinguish between the hemolytic patterns of different organisms

catalase

an enzyme that converts hydrogen per iodide into water and oxygen gas

if an organism contains catalase

it will bubble indicating the reaction has taken place

example of an organism that produce catalase

staphylococci

streptococci

example of an organism that doesn’t produce catalase

mannitol salt agar (MSA)

a selective and differential media that has a NaCl concentration of 7.5% not permitting the growth of a lot of bacteria

coliforms

organisms that can ferment lactose

paracolons

organisms that cannot ferment lactose

MacConkey agar

selective and differential medium which is selective for gram negative bacteria and contains blue salts, crystal violet, lactose, and pH indic. neutral red

eosin methylene blue levine agar (EMB)

selective and differential medium that is partially inhibitory for gram positive bacteria

appearance of lactose fermentation on MacMonkey agar

red

appearance of lactose fermenters on EMB agar

deep purple or blue black with a metallic green sheen from strong acid production

salmonella shigella (SS) agar

a selective and differential medium used to isolate & identify salmonella and shigella species from other bacteria

appearance of lactose fermentators on SS agar

red, but bacteria that also produce H2S will have a black center

Triple sugar iron agar (TSI)

allows for differentiation between bacteria :

1. based on their ability to ferment glucose, lactose, &/or sucrose

2. to produce gas from fermentation

3. their ability to reduce sulfur to hydrogen sulfide

glucose

sugars fermented during an alkaline slant reaction

glucose fermentation interpretation

sugar is depleted quickly and if bacterium can’t ferment lactose &/or sucrose, it will begin to utilize proteins in the agar. ammonia is produced raising the pH turning the slant alkaline w/ a yellow appearance

glucose, lactose, &/or sucrose

sugar fermented during an acidic TSI reaction

glucose, sucrose, and/or lactose fermentation interpretation

once glucose is depleted, the organism will ferment sucrose or lactose if available turning the slant acidic

mannitol salt

• pH indicator: phenol red

• inhibitors: 7.5% NaCl

• fermentable sugars: mannitol

• sodium thiosulfate and Fe++: negative

EMB

• pH indicator: eosin & methylene blue

• inhibitors: eosin & methylene blue

• fermentable sugars: lactose

• sodium thiosulfate and Fe++: negative

SS

• pH indicator: neutral red

• inhibitors: bile salt and brilliant green

• fermentable sugars:lactose

• sodium thiosulfate and Fe++: positive

MacConkey

• pH indicator: neutral red

• inhibitors: bile salt and crystal violet

• fermentable sugars: lactose

• sodium thiosulfate and Fe++: negative

TSI

• pH indicator: phenol red

• inhibitors: none

• fermentable sugars: glucose, lactose,& sucrose

• sodium thiosulfate and Fe++:

citrate

• pH indicator: bromothymol blue

• inhibitors: none

• fermentable sugars: none

• sodium thiosulfate and Fe++: negative

gram positive bacteria

• staphylococcus aureus

• staphylococcus epidermis

• streptococcus pyogenes

• streptococcus pneumonia

gram negative bacteria

• escherchia coli

• klebsiella pneumoniae

• salmonella enteric a

• yersinia enterocolitica

staphylococcus aureus traits

• gram positive

• cocci

• grape like clusters

• beta hemolytic

• catalase producer

• NaCl tolerant

• can ferment manitol (yellow)

staphylococcus epidermidis

• gram positive

• cocci

• grape like clusters

• gamma hemolytic

• catalase producer

• NaCl tolerant

• cannot ferment mannitol (red)

streptococcus pyogenes traits

• gram positive

• cocci

• chains or pairs

• beta hemolytic

• cannot produce catalase

• cannot tolerate NaCl

• can’t ferment mannitol (red)

streptococcus pneumoniae

• gram positive

• cocci

• chains or pairs

• alpha hemolytic

• can’t produce catalase

• can’t tolerate NaCl

• can’t ferment mannitol

e. coli traits

• gram negative

• rods

• can ferment lactose (yellow)

• can’t produce H2S

• can’t utilize citrate (green &/or no growth)

klebsiella pneumoniae traits

• gram negative

• rods

• can ferment lactose (yellow)

• can’t produce H2S

• can utilize citrate (blue)

salmonella enterica traits

• gram negative

• rods

• can’t ferment lactose (bright red TSI)

• can produce H2S (black dot)

• utilizes citrate (blue slant)

escherichia coli expected test results

• EMB: dark purple w metallic green sheen

• SS: mostly inhibited, pink colonies if present

• Mac: bright pink colonies (contains lactose, pH > 6.8)

• TSI: slant:acidic (y), butt: acidic (y), gas varies, no H2S

• Citrate: not utilized (green)

• MSA: inhibited

salmonella enterica test results

• EMB: colorless colonies and coloration of medium

• SS: colorless colonies w black center

• Mac: colorless colonies

• TSI: slant: alkaline (p), butt: acidic (y), no gas, produces H2S

• Citrate: utilizer (blue)

• MSA: inhibited

klebsiella pneumoniae test results

• EMB: dark purple w little to no green

• SS: mostly inhibited, pink colonies if inhibited

• Mac: bright pink colonies

• TSI: acidic slant (y), acidic butt, gas varies, and no H2S production

• Citrate: utilizer (blue slant)

• MSA: inhibited

yersinia enterocolticia test results

• EMB: color of medium w colorless colonies

• SS: colorless colonies

• Mac: colorless colonies

• TSI: alkaline slant, acidic butt, gas varies, no H2S production (may produce A/A result bc it ferments sucrose)

• Citrate: does not utilize (green slant)

• MSA: inhibited

streptococcus pneumoniae test results

BAP: alpha hemolytic (green halo)

Catalase: can’t produce

MSA: inhibited

EMB: inhibited

SS: inhibited

Mac: inhibited

streptococcus pyogenes test results

BAP: beta hemolytic (clear halo)

MSA: inhibited

Catalase: cannot produce

EMB: inhibited

SS: inhibited

Mac: inhibited

staphylococcus aureus test results

BAP: beta hemolytic (clear halo)

MSA: growth w yellow medium

Catalase: producer

EMB: inhibited

SS: inhibited

Mac: inhibited

staphylococcus epidermidis test results

BAP: gamma hemolytic (no change)

MSA: growth w no color change

Catalase: producer

EMB: inhibited

SS: inhibited

Mac: inhibited