Micro Exam 2 Flashcards

pure culture

mixed culture

aseptic/sterile techniques

aseptic transfer

sterile

contamination

broth

agar slants

meniscus

to isolate individual species from mixed culture so one can identify a specific organism

produces a flame with two cones (outer and inner), and the bottom knob adjusts the volume of gas (counter-clockwise increases the flame)

the top of the inner cone

the outer cone

inoculating loops, inoculating needles, sterile pipettes are also used, and sterile swabs

serological

Mohr pipette

sterilizing the countertop, flaming the loop/needle, use the lid of the petri dish to contain airborne, alcohol-flame forceps and use the sterilized tips to transfer material, and disinfect the countertop again

boiling droplets of microorganisms spray into the air when a loop or needle full of bacteria is thrust into a flame

flame loops and needles from the handle connection to the tip, so the bacteria are charred before incinerating them

or

wait for microbes to dry and flame sideways (slight angle, not completely sideways)

wear lab coat, closed-toed shoes, safety glasses, wash hands before leaving lab

we make a stock culture as a duplicate because the stock culture that we used for transfer is no longer sterile

the biggest difference is that eukaryotes have membrane-bound organelles, while prokaryotes do not

morphologies

arrangement

good job

cocci (pl.) coccus (s.) - spheres

bacilli (pl.) bacillus (s.) - rods

spirlla (pl.) spirillum (s.) - spirals

vibrios - slightly curved rods

coccobacilli - short rods

spirochetes - flexible spirals

diplo-

cocci and bacilli

strepto-

cocci and bacilli

tetrad; cocci

sarcina (cube); cocci

staphylo-

cocci

palisade and angular

diphtheroids

palisade

spores

Both are spiral but spirilla are more rigid and use polar flagella for motility whereas spirochetes are flexible and motility through axial filaments.

slightly curved rods

some bacterial species grow in a variety of shapes and are said to be pleomorphic

bacteria are unicellular, microscopic and prokaryotic organisms; most of them are devoid of chlorophyll

Cyanobacteria (blue green algae) are a group of gram negative photosynthetic (contain chlorophyll a) prokaryotes; live in extreme habitats; do not contain flagella

Akinete cyanobacteria

heterocyst cyanobacteria

cell dimension = ocular units x calibration...

low= 10 µm

high= 2.5 µm

oil= 1.0 µm

simple microscope

compound microscope

Total magnification= magnification by the objective lens X magnification
by the ocular lens

10x

4x

40x

10x

100x

40x

400x

100x

1000x

Using immersion oil between the specimen and the lens increases the numerical aperture (measure of a len's ability to "capture" light coming from the specimen and use it to make the image) that makes its limit of resolution smaller.

Oil causes a better resolution because the refractive index of oil is very similar to refractive index of glass; It increases better magnification by eliminating the transfer from glass to air

Resolving power: actual measurement of how far apart two points must be for
microscope to view them as separate.
Numerical aperture: a measure of a lens ability to "capture" light coming in fromspecimen and use to make an image.

the blue filter increases contrast between a specimen and its background;
better higher resolution

As magnification increases, the working distance decreases (Working distance is the distance between the objective lens and the specimen)

when observing samples at higher magnification, the microscopist has to be careful to not make the working distance completely squashed against the slide and lens

Parfocal: having corresponding focal points all in the same plane; having sets of objectives or eyepieces so mounted that they may be interchanged without varying the focus of the instrument (as a microscope) with which they are used

Advantage: allows more accurate focusing at maximum focal length and then
zooming back to a shorter focal length to compose the image

locate and explain the function

-carry microscope to workstation using both hands (one hand grasping the microscope's handle and the other supporting the microscope beneath its base)

-use lens paper to gently clean condenser and objective lens

-when putting away, wipe the oil lens with lens paper and alcohol; lower stage, return objective lens to the 4x power; put bag back over and put back in storage spot

As magnification increases size of field view decreases. The field of view is how much of your subject you see through the lens.

One might not be able to see everything he/she needs to see with the higher power, so the mechanical stage would have to be moved to see different areas of specimen in the higher power

The optics of the microscope inverts the image (upside down); example was the "e" slide

okay

Human blood cells ~12 um
Yeast ~3-4 um
Bacilli ~0.2-2.0 um
Cocci ~1.0-2.0 um

- distance through which you can move the specimen and still have it remain in focus
- the thread exercise; To determine the relative position of all 3 colored threads, you must raise and lower your objective lens slowly (known as "focusing up and down") and observe the order in which the threads go in and out of focus. As your objective lens approaches the slide, the first thread to come into focus is on the top.

0.1 micometer (um) or 100 nanometers (nm)

the distance between the objective lens and specimen

increases

left

away from you

Because the image only goes through one ocular to reach both eyes

calculate for limit of resolution

D = wavelength/(NA object + NA condensor) visible light = 300-700 nm

limited resolution decreased if wavelength decreased

if wavelengths are small = smaller limit resolution

by closing the iris diaphragm, it closes the peripheral light giving it a better image

even though the specimens may be very small, they still have depth

-for streak plates (isolation of microbes to identify and create a pure culture)

-for spread plates (to create a uniform lawn)

-often used for isolating individual species from a mixed culture

-counting the number of cells in a diluted sample

Prepare new stocks by taking sample from old stock about to be used. Fish tail spread onto new slant using sterile inoculating loop and store at 25°-35°C depending on organism to be grown. Do not tighten cap all the way.

sterile plates are done by using sterile agar broth, flamed at the mouth of the tube right after opening, poured with the petri plate lid only slightly open and quickly closed, flame the empty tube and screw back on the lid

if the plate has been refrigerated this will prevent cold sensitive bacteria from shock or death; will evaporate any excess condensation on the media

make sure they are sterilized and have no unwanted growth before growing microbes

air, lip of tube

A mixed culture contains two or more species of microbe;
A pure culture contains a single species

the identification process of an unknown microbe relies on obtaining a pure culture of that organism

the streak plate method produces individual colonies on an agar plate, then a portion of an isolated colony then may be transferred to a sterile medium to start a pure culture

to avoid condensation disturbance of growth as well as rehydrate bacterial colonies

concentration gradient

confluent growth

quadrant method

inoculum

agar deep

colony

colony forming unit (CFU)

sterilized Inoculation loop used to drag bacterial sample through 4 quadrants (0,1,2,3) for the purpose of isolating individual colonies or pure cultures

Swab used to create a uniform lawn of bacteria for the purpose of seeing response to certain treatments

Grading rubric involves
Any evidence of bacteria? Is it fungal or bacterial
Where is contamination?
Using location as an indicator, what might the source of this contamination be?
Did you obtain isolated colonies? If so, in which quadrant did you have isolated colonies? Approximately how many well-isolated colonies are there?
What factors determine the size of a colony growing on a streak plate?
A Streak plate was made and observation of the results shows

Assumption is that colonies can be isolated or even lawn can be made.
Limitation is due to technique

streak plates: a method of bacterial isolation

spread plates: used for antibiotic and disinfectant testing

technique, nutrients, waste products, and antibiotics could all cause smaller colony formation

it is a gel at room temperature and most microbial species cannot break it down for food. It provides a growing matrix for microbial species through microscopic channels liquid filled channels that allow nutrients to diffuse through as food for microbes
Agar used to solidify (somewhat) media for growing organisms. Does not feed microbes. Nutrients must be added

Large inoculum would need larger quadrants and less dips into previous quad to spread out colonies more

turbidity

the McFarland standard

spread plates must be consistent to form a proper bacterial lawn to be used for testing; a known and consistent concentration of bacteria is spread on all plates

we do not standardize quadrant streaks because consistency is not the goal, isolation is the goal.

1. color
2. size
3. shape
4. texture

cation

anion

solvent

chromogen

chromophore

auxochrome

An acidic dye carries a negative charge because it is missing hydrogen; leaves the cell negatively charged and stains the background, repelling the bacteria

A basic dye carries a positive charge because it has either gained hydrogen or lost a hydroxide; it is attracted to the negatively charged cell

the concentration of the bacteria is too high, so serial dilution takes place to help isolate the bacterias on the slide better.

excess water left on the slide will boil during the fixing stage, causing most microbes present to rupture and become airborne

Basic stains are heat fixed.

Heat fixing kills bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible under the microscope

this can make the cell appear larger than it really is, with a simple stain it might be okay but with gram staining it would ruin it

Overstaining the bacterial smear may cause the cell wall disruption or totally destroy the cell wall which results in the loss of true morphological characteristics of the bacterial cell

The cells may lose the stain when washed with alcohol or water which causes a problem in identifying the cell
In Gram staining if it is not stained properly with the Methylene Blue then it will lose the stain when washed and will counter stain with Safranin and cause false Gram negatives

Cocci, with their low surface to volume ratio are less efficient at exchange with the environment than rods, but are at an advantage in a dry environment where they lose water dehydrate more slowly than rods

Organisms with high surface to volume ratio (rods,spirilla) often survive better in moist environments where their ability to exchange materials with their surroundings is an asset for nutrient acquisition of water loss is not a concern

Congo red and Nigrosin (India Ink)

the negative stain uses a dye solution in which the chromogen is acidic and carries a negative charge

the negative charge on the bacterial surface repels the negatively charged chromogen, so the cell remains unstained against a colored background.

negative stains cannot be heat fixed because they are too delicate to withstand heat-fixing; the morphology would be destroyed and size would shrink immensely

-Gently draw a second slide across the surface of the first until it contacts the drop so that the drop will spread across the edge of the top slide
-Push the top slide to the left along the entire surface of the bottom slide

-used to determine morphology and cellular arrangement in bacteria that are too delicate to withstand heat-fixing

-to avoid distorting of the cell

capsules

Excreted by cell to form clear, gelatinous, protective layer

Background will appear darker (negative stain), then a clear barrier surrounding the stained cell

the negative stain doesn't colorize the cells in the smear because the negative stain's chromogen, which carries a negative charge, is repelled by the negative charge on the cell's surface

therefore the stain colors only the background, allowing visibility of the cell and/or its capsule

Eosin--is acidic and acts as a negative stain
Methylene blue--is basic

The smears background would turn out red while the cells would turn out blue.

serum acts as glue to hold it to the slides

capsules help bacteria stick to teeth

internally, capsules help prevent phagocytosis

The Gram stain was developed in 1884 by Hans Christian Gram.

It is one of the most useful staining procedures because it classifies bacteria into two large groups: gram positive and gram negative

primary stain: crystal violet
mordant: iodine
decolorizer: alcohol or acetone
counterstain: safranin

The crystal violet stains all the cells

The iodine forms a crystal violet-iodine complex with the crystal violet to keep it in the cell, called a mordant

The decolorizer (alcohol or acetone) allows the crystal violet-iodine complex to leave the gram-negative cells

The counterstain (Safranin) stains the gram-negative cells pinkish red

Gram negative cell walls have a higher lipid content (because of the outer membrane) and a thinker peptidoglycan later than Gram-positive cell walls

the alcohol/acetone extracts the lipid, making the gram-negative wall more porous and incapable of retaining the crystal violet-iodine complex, thereby decolorizing itt

the thicker peptidoglycan and greater degree of cross-linking (because of techoic acids) trap the crystal violet-iodine complex more effectively, making the Gram-positive wall less susceptible to decolorization

the alcohol/acetone extracts the lipid, making the gram-negative wall more porous and incapable of retaining the crystal violet-iodine complex, thereby decolorizing it

the thicker peptidoglycan and greater degree of cross-linking (because of techoic acids) trap the crystal violet-iodine complex more effectively, making the Gram-positive wall less susceptible to decolorization

Gram-nonreactive bacteria are bacteria without a cell wall such as Mycoplasma species, or with cell walls resistant to this staining procedure such as Mycobacterium species

Cultures 24 hours old or less should be used, or else they will lose their ability to retain the crystal violet-iodine complex

The stain can be over-decolorized by leaving the alcohol on too long, creating reddish gram-positive cells

The stain can also be under-decolorized, creating purple gram-negative cells

Inconsistency in preparation of the emulsification can also occur

The Crystal Violet would be leeched out of everything, because the Iodine is what cross-links with the Crystal Violet. And that cross-link is what help the stain to stay within the cell during decolorization. When the counterstain, Safranin, is applied everything will end up pink and appear Gram-negative.

Everything will look purple at the end of the staining procedure

You would not be able to see the Gram-negative cells. The Gram-positive would be the only cells that are colored and therefore visible. Gram-negative would be clear and therefore not visible

a) Everything would appear purple at the end.
b) During decolorization the Safranin would be leeched out, the Mordant; Iodine, will only cross-link with the Crystal Violet.

a) The properties of the stains themselves, the Crystal Violet will cross-link with the mordant, Gram Iodine and the Safranin will not cross-link.
b) The Gram staining procedure is a contributing factor.

No, only Bacteria are Gram-positive or Gram-negative.

Human cells do not have cell walls or Peptidoglycan (PDG). The cells could take either color stain.

I would suggest that she make sure she is using new cultures, because if she is using old cultures then they lose their ability to retain the Crystal Violet.

they have mycolic acid in the cell walls of the organisms; this is the cytological basis for the acid-fast differential stain

it is waxy substance that gives acid-fast cells a higher affinity (bond with) for the primary stain (carbolfuchsin) and resistance to decolorization by an acid alcohol solution

Both: primary stain for 5 minutes, decolorize, counter stain for 1 minute
ZN: acid fast are reddish purple, non acid fast are blue
K: acid fast are reddish purple, non acid fast are green

Mycobacterium, Nocardia, Cryptosporidium, Isospora

ZN and K:
carbolfuchsin- primary stain
Acid alcohol- decolorizer
Methylene blue- counterstain

the only difference is that ZN uses heat-fixing (steam),

K method also uses a slightly more lipid-soluble and concentrated carbolfuchsin; brilliant green or methylene blue can be used as the counterstain

Z uses steam to stain, K uses a more lipid-soluble carbolfuchsin stain

95% ethanol, 3% HCl

the mycolic acid in their cell walls resist the decolorization; the primary stain carbolfuchsin is lipid soluble and penetrates the waxy cell wall

their waxy cell walls repel typical aqueous stains

give the cell a higher affinity (liking) for the stain and help it resist decolorization

to help the "slippery" acid-fast cells adhere to the slide

help drive the primary stain into the waxy cell walls of these difficult-to-stain cells

Yes, they are initially stained by carbolfuchsin but the decolorizing step, acid alcohol, removes stain from acid fast negative cells while acid fast positive cells retain the stain

Acid fastness is a characteristic that is shared by just a few organisms. Many bacterial cells are easily stained with simple stains or using the Gram stain.

-acid fast is useful when acid fast positive bacteria are suspected

1. Begin with a heat-fixed emulsion
2. Cover the smear with a strip of bibulous paper. Apply Malachite Green stain. Steam for 7-10 min.Keep the paper moist with stain.
3. Remove the paper and dispose of it. Rinse the slide
4. Counterstain with Safranin stain for 1 min. Rinse
5. Blot dry with bibulous paper. Observe under oil immersion.

ta-da

1. all cells are stained with Malachite green using steam to force the stain into the spore, if present
2. Decolorized with water to remove stain from cells, but not spores
3. Safranin is used to counterstain vegetative and spore mother cells

terminal: at the end of the cell
subterminal: between the middle and end of the cell
central: in the middle of the cell

cyptobiotic state

sporangium

keratin

spore mother cell

location in cell
shape
size relative to cell

Bacillus and Clostridium

Sporosarcina

1) endospore-forming rod that causes diarrhea and pseudomembranous colitis. This organism is the leading cause of antibiotic-associated diarrhea (AAD). This organism's nickname is "C-diff." ..... Clostridium difficile

Young cultures of spore-forming microbes may not demonstrate any endospores because the vegetative cells may not have been subjected to sufficient stress to stimulate sporulation

positive results: shows cell has ability to produce spores, which narrows down indentification of bacteria to a few genera of bacteria

negative reults- non spore producing bacteria

Endospore stain is a differential staining procedure that allows to see both spores and vegetative cells, thus including separate -ve control consisting of only vegetative cells is not required

unstained objects can be something other than an endospore, such as lipid granules

serial dilution

agar pour (deep)

too numerous to count

too few to count

countable plate

lenticulate

colony forming unit (CFU)

heterotrophic plate count

standard methods agar

aka

plate count agar

a colony-forming unit (CFU) can be any number of cells, whether a single cell or a group of two or more cells; a single cell is only one single cell

When 0.1 mL of a 10^-4 solution is transferred to a plate, what is the volume of the sample in the plate?
-0.1 mL x 10^-4 = 10^-5 mL

<30 colonies, statistically unreliable TFTC
>300 colonies, too many, TMTC

should at least produces 30-300, 130 is countable

the number of colonies is equal to the number of bacteria per gram of meat transferred

x colonies

_________

1x10^{- x} dilution

so...... (x colonies)(1x10^x)

-for the Health and Safety tests (must be under 10⁶ bacteria/ gram of meat)

-Medical microbiologist manipulate population density for use for standardized testing

-To measure the effect of varying nutritional or environmental conditions.

-Industrial microbiologist maintain populations at optimum levels in products including enzymes, antibiotics, beer and wine

must be under 10⁶ bacteria/ gram of meat

-one limitation is that only bacteria capable of growing in the culture medium under the environmental conditions provided will be counted

intended= 1x10^-2
actual= 4.8x10^-2

The original cell density (OCD) = 1.54 x 10⁷ CFU/mL

1 mL was used to inoculate this plate

The original cell density (OCD) = 6.2 x 10⁸ CFU/mL

1 mL was used to inoculate this plate

to determine number of microbes in 1g of food

if greater than 10⁶ it may be hazardous to consume

transient - temporary microorganisms that can be washed off hands with soap or other detergent

resident - long-term flora that generally inhabit hair follicles and sebaceous glands of skin that remain even after thoroughly washing, scrubbing, and disinfecting hands

Detergent is any cleansing solution, including soap, to remove bacteria embedded in fats and oils

Soap is formed when fats are heated in the presence of a name such as sodium hydroxide (NaOH)

They both decrease surface tension so that microbes can be more easily removed

In greasy water, the hydrocarbon end of the soap molecule is soluble in oil and the carboxylate end is soluble in water.

A chemical link forms between oil and water. Then the oily ends of the molecules dissolve together. Now the greasy oil is chemically combined with water; it can be carried away in the liquid.

Hard water is water containing mineral salts. When soap is used with hard water, magnesium and calcium salts react with soapy oil droplets, forming a lighter than water precipitate, scum. This scum, with its load of grease and bacteria, adheres to whatever is being washed.

Antiseptic detergents work better than regular soap

It's important to have control plates for this experiment so you can distinguish between bacteria that possibly came from the substance/object you were using and the resident/transient bacteria on the hand.

It was an attempt to spread out the possible different bacteria on the hand

yippee!