Microbio Lab Midterm exam

resolving power determined by

wavelength and aperture

immersion oil

used on 100x lens, results in less bending of light and better image quality

* has higher refractive index than air
* has better resolution

culture medium

uses soluble low molecular weight substances that are derived from complex nutrients broken down by enzymes. Used to grow microorganisms

agar

an extract of seaweed, a complex carbohydrate composed mainly of galactose

* liquifies at 100C
* solidifies at 40C

Antonie van Leeuwenhoek

first person to observe microbes, called them wee little beasties

resolving power

relates to how well a lens allows two objects to be seen as distinct and separate

total magnification

objective lens x ocular lens

phase contrast microscope

uses refraction and interference to see high contrast high resolution images

fluorescence microscope

uses fluorescent stains to produce image

confocal microscope

uses laser to scan plane and produce numerous 2 dimensional high resolution images at various depths that can be constructed into 3D image

* can be used for biofilms

two photon microscope

uses scanning technique, fluorochromes and long wavelength lights

transmission microscope

uses electron beams that pass through a specimen to visualize small images

scanning microscope

uses electron beam to visualize surfaces to create 3D image

petri dishes are incubated in an

inverted position

aseptic technique

practices and procedures used to prevent contamination from pathogens and minimize risk of infection

* handwashing, sterilization, keeping flasks closed

subculturing

transferring of microorganisms into fresh medium from its stock culture

streak plate

used to dilute bacteria until single cells are separated from one another for identification

* isolation of distinct colonies

filiform growth

continuous threadlike growth with smooth edges

echinulate growth

continuous growth with irregular edges

beaded growth

nonconfluent to semiconfluent colonies

effuse growth

thin spreading growth

arborescent growth

treelike growth

rhizoid growth

rootlike growth

spread plate

requires that a previously diluted mixture of microorganisms be used

stain dye

defined as an organic compound containing a benzene ring plus a chromophore and auxochrome group

chromogen

benzene + chromophore

* colored compound but not stain

auxochrome

enables chromogen to form salts and bind to fibers or tissues

acidic stains

anionic (negative charge), has strong affinity for positive parts of cell

basic stains

cationic (positive charge) that has a strong affinity for negatively charged parts of cell such as cell walls

bacterial smears

done before staining procedure, should appear translucent/semi transparent

* require heat fixation to fix bacterial proteins to glass slide before staining

gram positive

bacteria that has thick peptidoglycan layer and is stained with primary stain

* purple appearance

gram negative

bacteria that has thin peptidoglycan layer surrounded by outer lipid containing layers

* stains with counterstain
* dyed with red counterstain

gram staining

1. primary stain (crystal violet)
2. mordant - gram’s iodine
3. counterstain - alcohol wash


1. counterstain - red safranin

acid fast staining (ziehl neelsen method)

used to detect mycobacterium that have mycolic acid (waxy polymer that is less permeable)

acid fast staining steps

1. primary stain - carbolfuchsin
2. steam
3. decolorizing agent - acid alcohol
4. counterstain - methylene blue

spore stain (schaeffer fulton method)

used to detect endospores from bacillus and clostridium

* exist as metabolically active vegetative cells
* exist as metabolically inactive spores

spore stain steps

1. primary stain (malachite green)
2. steam heating
3. decolorizing agent - water
4. counterstain - safranin

clostridium endospores

have slight bulge around spore

bacillus endospores

do not have bulge around endospore

general growth media

non specific media configured to cultivate a wide range of microorganisms without many restrictions

selective media

specifically configured to restrict growth of particular organisms while allowing others to grow

* bile salts, crystal violet, antibiotic

differential media

contains chemical compounds that cause an observable change in the medium or the medium surrounding the bacteria when a particular biochemical reaction occurs

autotrophs

can be cultivated in a medium consisting of inorganic compounds, use inorganic carbon in form of CO2

heterotrophs

cannot be cultivated in a medium consisting of inorganic compounds, must be supplied with organic nutrients such as glucose

mannitol salt agar

selective for staphylococcus, differential for staph aureus

* staph aureus + mannitol = fermented acid product
* phenol red → yellow halo

blood agar

selective for pathogenic streptococci

* ability for strep to cause hemolysis
* alpha (partial), beta (full), gamma (none)
* tryptic soy agar + sheep blood
* also looks for enterococci and aerococci

macconkey agar

selective for gram negative enterobacteria

* contains bile salts/crystal violet to inhibit gram +
* distinguishes coliforms from non coliforms
* contains lactose for coliform to ferment
* neutral red indicator = red color when positive

Eosin Methylene Blue

selective for gram negative enterobacteria

* contains eosin and methylene blue dye to inhibit growth of gram +
* distinguishes coliforms from non coliforms
* methylene blue → red if acidic (fermentation)
* methylene blue + eosin = green for e coli

phenylethyl alcohol agar

selective for gram positive organisms

* inhibits growth of gram negative

psychrophile

bacteria that grows between -5 to 20 C

all grow between 0 and 5 C

mesophile

bacteria that grows at 20 to 45 C

psychrotolerant

optimum growth temperature is 20-40C

can grow at 0C

thermophiles

bacteria that grows at 35C and above

facultative thermophile

organisms that grow at 37C but optimally at 45-60C

obligate thermophile

bacteria that grows only at temps above 50C

aerobes

requires presence of atmospheric oxygen, uses oxygen as final electron acceptor

microaerophiles

require limited amounts of atmospheric oxygen for growth, excess oxygen results in death

obligate anaerobes

requires absence of free oxygen for growth, presence of oxygen results in formation of toxic metabolites

aerotolerant anaerobes

produce catalase and are not killed in presence of oxygen, do not use oxygen as final electron acceptor

facultative anaerobes

can grow in presence of absence of oxygen, prefer to use oxygen for aerobic respiration

lag phase

cells transition from maintenance metabolism to growth metabolism

* enzymes for cell growth are synthesized

log phase

cells are now in growth metabolism and cell number doubles regularly until max is reached

* length of this phase depends on organism and medium composition

stationary phase

cells transition to maintenance metabolism and number of cells undergoing division is equal to number of cells that are dying

* toxic end products begin to accumulate

decline phase

waste products build up and cells in culture die at rapid rate

growth rate formula

g = t/n

* g : generation time
* t : time of exponential growth
* n : number of generations
* \*\*\* equation used only for log phase

extracellular enzymes

act on substances outside of cell, mainly hydrolytic that reduce high molecular weight substances to low molecular weight substances for entry into cell

* starch hydrolysis
* lipid hydrolysis
* gelatin hydrolysis

intracellular enzymes

function inside cell and mainly responsible for synthesis of new material for cell energy

* basis of cell metabolism

starch hydrolysis

starch is polymer of glucose molecules linked by glycosidic bonds

* starch + amylase enzymes = maltose
* starch agar flooded with iodine
* clear zone = positive
* blue black + no clear zone = negative

lipid hydrolysis

lipids + lipase + H2O → glycerol + fatty acids

* tributyrin agar (lipid substrate)
* clear area around growth indicates hydrolysis

gelatin hydrolysis

gelatin + gelatinase → amino acid

* gelatin deep inoculated with stab inoculation
* cultures that remain liquid after refrigeration = pos
* cultures that remain solid = negative

fermentation

the anaerobic breakdown of substrates like carbohydrates and alcohols with the production of organic acids and sometimes gases such as H2 or CO2

carbohydrate fermentation

includes nutrient broth with carbohydrate, pH indicator phenol red, inverted durham tube

* yellow coloration = fermentation
* yellow color + gas bubble = fermentation and gas
* no color change = no fermentation

IMViC test series

used to differentiate enterobacteria found in intestinal tract

* pathogens = salmonella, shigella
* occasional pathogens = proteus, klebsiella
* non pathogenic = E coli, enterobacter

indole production test

tryptophan + tryptophanase = indole + ammonia + pyruvic acid

* SIM agar contains tryptophan
* Kovac’s reagent added
* red layer = positive

methyl red test

differentiates E. coli from K. aerogenes

* glucose → organic acid end products
* indicator methyl red added
* red color = pH 4, positive E coli
* yellow color = pH 6, positive K aerogenes

Voges-Proskauer test

tests for ability to produce nonacidic/neutral end products from organic acid products of glucose metabolism

* Barritt’s reagent added and reacts with acetylmethylcarbinol (AMC)
* rose colored product = positive

citrate utilization test

tests ability of bacteria to use citrate as sole carbon source

* bromothymol blue indicator
* blue color = positive (in presence of alkaline sodium carbonate)
* green color = negative

citrate + citrase → oxalacetic acid + acetate → pyruvic acid + CO2

* CO2 + Na + H2O → sodium carbonate (alkaline)

hydrogen sulfide test

tests for ability to produce H2S from amino acids or inorganic compounds

* SIM medium contains sodium thiosulfate
* sodium thiosulfate + thiosulfate reductase → sulfite + hydrogen sulfide gas
* ferrous sulfate indicator
* combines with H2S gas to form black precipitate (ferrous sulfide)
* FeSO3 + H2S → FeS

urease test

tests for presence of enzyme urease which catalyzes hydrolysis of urea into CO2 and ammonia (alkaline)

* urea + urease → CO2 + H2O + NH3
* phenol red indicator
* turns pink in alkaline pH (positive result)

nitrate reduction test

tests for ability to reduce nitrates to nitrites

* NO3 + 2H + 2e + nitrate reductase → NO2 + H20
* nitrites may be further reduced to ammonia or nitrogen
* test solutions A and B indicators
* red = positive
* zinc dust
* red color = negative
* no color change = positive

catalase test

tests for presence of enzyme catalase which degrades hydrogen peroxide

* T soy plate
* if mixture bubbles upon addition of H2O2, O2 is produced and positive for catalase
* 2H2O2 + catalase → 2H20 + O2

oxidase test

cytochrome oxidase catalyzes oxidation of reduced cytochrome by oxygen resulting in water of hydrogen peroxide

* reagent p-aminomethylanaline oxalate oxidized in presence of cytochrome oxidase
* t soy plate becomes pink,maroon then black when positive
* pink or no color change = negative