free of living microbes (unable to reproduce)

sterile

the process of something becoming impure or unclean

contamination

a fluid culture medium used to grow microbes when fresh cultures or large numbers of cells are required; also used in microbial identification

broth

used to grow stock cultures in a tube that can be refrigerated after incubation and maintained for several weeks

agar slants

the bottom of this is used to measure the volume of a liquid

meniscus

when must a microbiologist use a pure culture?

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

what are the characteristics of a properly adjusted bunsen burner flame?

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

sterilization of inoculating instruments is done in the hottest part of the flame - ______________________________, though heat-fixing bacterial smears on slides and incinerating the mouths of open glassware items may be done in _________________________

the top of the inner cone

the outer cone

what are the instruments that microbiologists most often use to transfer microorganisms?

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

this pipette style is calibrated to deliver (TD) its volume by completely draining it and blowing out the last drop (the measurement markings end at the very end of the end of of the tip)

serological

this style of pipette is not graduated, so fluid must be stopped at a calibration line; stopping the fluid beyond the last line on this pipette results in an unknown volume being dispensed (the measurement markings end before the very end of the tip)

Mohr pipette

identify aseptic technique procedures that are performed before, during and after working with microorganisms

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

how are aerosols formed?

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

how do you prevent aerosols from forming?

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)

explain how to protect yourself from laboratory-aquired infection

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

understand the purpose of stock cultures and why a new stock culture is always made as the first inoculating from a stock culture tube

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

distinguish between prokaryotic and eukaryotic cells

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

bacterial cells come in a variety of __________________ (shapes) and _____________________ (the number of planes in which division occurs and whether the cells separate after division)

morphologies

arrangement

Look over the morphologies (common shapes) and arrangements of prokaryotic cells on the microscope worksheet

good job

the different morphologies of cells include (6 of them):

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

the cell arrangement prefix for pairs of cells is... and these two cell morphologies can form it

diplo-

cocci and bacilli

the cell arrangement prefix for chains of cells is... and these two cell morphologies can form it

strepto-

cocci and bacilli

the cell arrangement for second division in a plane perpendicular to the first is called... and this arrangement is only formed with this cell morphology

tetrad; cocci

the cell arrangement for a third division plane perpendicular to the other two which produces a cube-shaped arrangement of eight cells is called... and this arrangement is only formed with this cell morphology

sarcina (cube); cocci

the prefix is cell arrangement that has irregular division planes, or is a cluster of cells is called... and only this cell morphology can form it

staphylo-

cocci

bacilli can also form __________ and ___________ cell arrangements

palisade and angular

any bacterium of a genus that include the diphtheria bacillus, especially one that does not cause disease; a large group of nonpathogenic bacteria gram-positive bacteria that resemble corynebacterium

diphtheroids

this arrangement is a stacking of rod-shaped cells side-by-side

palisade

A state of "suspended animation" that some bacteria can adopt when conditions are not ideal for growth

spores

differentiate spirilla and spirochetes by description and observation

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

what is a vibrio?

slightly curved rods

what does pleomorphic mean?

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

differentiate heterotrophic eubacteria (bacteria) and cyanobacteria; recognize examples of each

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

__________ are thick walled spores; they have protective layers, like an endospore; It is usually formed after a heterocyst due to harsh environmental conditions

Akinete cyanobacteria

this cell's sole function is to fix nitrogen when ammonia, nitrate, and other fixed forms of nitrogen are unavailable for a cyanobacteria

heterocyst cyanobacteria

given the calibration factor of an ocular micrometer, determine the size of an object viewed through a microscope...

at low power, each division in the ocular micrometer is ______ (include unit)

at high power, each division in the ocular micrometer is ______

at oil, each division in the ocular micrometer is ______

cell dimension = ocular units x calibration...

low= 10 µm

high= 2.5 µm

oil= 1.0 µm

this microscope consists of a single lens to enlarge an object

simple microscope

this microscope includes an eyepiece and one or more objectives where
the eyepiece enlarges the resolved image created by the objectives.

compound microscope

given the magnifications of an ocular and an objective lens, calculate the total magnification of a compound microscope

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

how much magnification is given by the ocular (eyepiece) lens?

10x

magnification of lowest power is ______

plus ocular = total magnification of: _______

4x

40x

magnification of low power is ______

plus ocular = total magnification of: _______

10x

100x

magnification of high power is ______

plus ocular = total magnification of: _______

40x

400x

magnification of oil immersion is ______

plus ocular = total magnification of: _______

100x

1000x

explain how the use of immersion oil allows the microscopist to obtain greater resolution

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

describe the relationship between resolving power, numerical aperture, and the wavelength of light

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.

Explain why a blue filter is used beneath the condenser of our microscopes (blue light is shorter wavelengths)

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

describe the change in working distance as magnification increases; what does this mean in practical terms with regard to observing samples at higher magnification?

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

evaluate the advantage to the microscopist of parfocality.. what does parfocal mean?

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 name the parts of your microscope; explain the function of each part**

locate and explain the function

list rules for the proper care of your microscope; know the checklist for putting a microscope away

-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

describe the change in the size of the field of view as magnification increases; understand what that means in practical terms as one increases magnification when viewing a slide

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

recognize how the optics of the microscope change the orientation of the image seen when compared to the orientation on the slide

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

observe bacteria using oil immersion. Be able to identify using correct terminology the shape and arrangement of stained bacteria

okay

compare the size of human blood cells with yeast cells and typical bacilli and cocci

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

understand what is meant by the term "depth of field;" how was this observed in the cork and threads' fibers slides?

- 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.

With compound light microscope we are able to see objects as small as

0.1 micometer (um) or 100 nanometers (nm)

Working distance is

the distance between the objective lens and specimen

As working force decreases the magnification

increases

when the stage moves to the right, the image moves to the...

left

when the stage moves toward you, the image moves...

away from you

Why aren't the magnifications of both ocular lenses of binocular microscope used to calculate total magnification?

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

Assuming that all other variables remain constant, explain why light of shorter wavelengths will produce a clearer image that light of longer wavelengths

calculate for limit of resolution

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

limited resolution decreased if wavelength decreased

why is wavelength the main limiting factor on limit of resolution in light microscopy?

if wavelengths are small = smaller limit resolution

Why should closing the iris diaphragm improve your ability to determine thread order?

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

what does the colored-threads slide demonstrate about specimens you will be observing later in the class?

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

list two common uses for poured plates

-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

understand the process of preparing and storing stock cultures

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.

describe and practice the process of preparing sterile plates

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

understand why, when preparing plated media, it would be useful to incubate the plates before using them

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

identify common sources of contaminants when preparing plated media

air, lip of tube

distinguish between a mixed culture and a pure culture

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

explain why a microbiologist would need pure cultures

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

understand why plates are labeled on the bottom of the dish and inverted for incubation and storage

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

decreasing quantity

concentration gradient

growth of culture over entire surface of media (individual colonies are indistinguishable)

confluent growth

method in streak plating to obtain a pure culture. The petri dish is labeled on the bottom in four quadrants (0, I, II, III). Initial inoculum is spread in 0 quadrant. Each following spread will be taken from previous quadrant after sterilization of inoculating instrument, and each successive quadrant should exhibit less dense microbial growth with isolated colonies present in quadrant III and possibly II

quadrant method

sample being introduced to sterile culture media

inoculum

tube with solidified agar perpendicular to opening, as opposed to the slant. Used to study the gas requirements of microorganisms (aerobic vs anaerobic)

agar deep

a macroscopically visible cluster of identical microorganisms

colony

this encompasses colonies that may arise from an individual cell, a pair of cells, chains, or clusters

colony forming unit (CFU)

give the goal for preparing a streak plate and describe the method for preparing a plate

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

Give the goal for preparing spread plates and describe how they are made

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

Prepare streak plates and spread plates, avoiding common pitfalls and contamination. (Note: The streak plate method used in this laboratory is provided on Bb in the Supplement (Ex 13).

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

understand the assumptions and limitations of these procedures

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

Identify use(s) for streak plates and spread plates

streak plates: a method of bacterial isolation

spread plates: used for antibiotic and disinfectant testing

Understand factors that will affect the size of colonies on plates

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

understand the purpose for using agar in biological media

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

Describe how the probable size of an inoculum will affect the way in which a streak plate will be prepared. (eg. size of the quadrants, number of “dips” into the previous quadrant.)

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

this is a measure of how cloudy or hazy a solution is as a result of bacterial growth

turbidity

what does the bacterial solution we made get compared to?

the McFarland standard

Why do we need to standardize the inoculum on a spread plate? Why don't we do this for a quadrant streak?

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.

List four characteristics of microbial morphology as identified in the lab book

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

positively charged ion

cation

negatively charged ion

anion

stains are solution consisting of a __________ (usually water or ethanol) and a colored molecule (often a benzene derivative), the ___________

solvent

chromogen

the portion of the chromogen that gives it its color

chromophore

the charged portion of a chromogen that allows it to act as a dye through ionic or covalent bonds between the chromogen and the cell

auxochrome

differentiate between basic and acidic dyes and understand how they work.

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

Understand why, when using solid media, the inoculum is serially diluted on the slide

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

Know the purpose of air-drying (or gently warming) smears

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

Know the purpose and effects of heat fixing a slide

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

What are some consequences of leaving a stain on a bacterial smear too long (over-staining)?

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

What are some consequences of not leaving a stain on a bacterial smear long enough (under-staining)?

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

Consider a coccus and a rod of equal volume, which is more likely to survive in a dry environment?

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

Consider a coccus and rod of equal volume, which is more likely to survive in a moist environment?

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

Identify two common negative stains:

Congo red and Nigrosin (India Ink)

Explain how a negative stain interacts with bacteria and the result obtained

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.

Understand why slides prepared with negative stains are not heat fixed

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

Understand the meaning of the rule: “draw the medium, do not push it” when making negative stained slides

-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

Identify two reasons for using a negative stain

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

-to avoid distorting of the cell

there are composed of mucoid polysaccharides or polypeptides that repel most stains because of their neutral charge

capsules

Know what a bacterial capsule is and how to demonstrate its presence by staining

Excreted by cell to form clear, gelatinous, protective layer

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

why doesn't a negative stain colorize the cells in the smear?

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 a red stain and methylene blue is blue. What should be the result of staining a bacterial smear with a misture of eosin and methylene blue?

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.

Capsules are neutrally charged. this being the case, what is the purpose of emulsifying the sample in serum in this staining procedure?

serum acts as glue to hold it to the slides

Some oral bacteria produce an extracellular "capsule." Of what benefit is a capsule to these cells?

capsules help bacteria stick to teeth

internally, capsules help prevent phagocytosis

Summarize the history and importance of the Gram stain

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

Identify the primary stain, mordant, decolorizer and counterstain used in the Gram staining procedure

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

Identify the purpose of each reagent of the Gram staining procedure

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

Describe and compare cell wall structure of gram-positive and gram-negative organisms

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

Understand how the differing wall structures are affected by the decolorization step of the Gram stain

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

Identify the characteristics gram-nonreactive bacteria and those bacteria for whom the Gram stain is not appropriate; give examples

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

Identify the variables which can affect the results of a Gram stain and understand how to limit the effects of these variables

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

predict the effect on Gram-positive and Gram-negative cells of the following "mistakes" made when performing a Gram stain. consider each mistake independently

1) failure to add the iodine

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.

predict the effect on Gram-positive and Gram-negative cells of the following "mistakes" made when performing a Gram stain. consider each mistake independently

1) failure to apply the decolorizer

Everything will look purple at the end of the staining procedure

predict the effect on Gram-positive and Gram-negative cells of the following "mistakes" made when performing a Gram stain. consider each mistake independently

1) failure to apply the safranin

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

predict the effect on Gram-positive and Gram-negative cells of the following "mistakes" made when performing a Gram stain. consider each mistake independently

1) reversal of crystal violet and safranin stains

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.

Both Crystal Violet and Safranin are basic stains and may be used to do simple stains on gram positive and gram-negative cells. This being the case, explain how they stain different cell types in the Gram stain.

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.

If you saw large eukaryotic cells in the preparation made from your gumline, they were most likely your own epithelial cells.

1) Are you gram positive or gram negative?

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

If you saw large eukaryotic cells in the preparation made from your gumline, they were most likely your own epithelial cells.

2) What is it about your cell structure that would allow you to predict this result?

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

One of your lab partners has followed the recommended procedure of running Gram-positive and Gram-negative control organisms on her Gram stain of an unknown species. Her choices of controls were Escherichia coli (gram-negative) and Bacillus subtilis (gram-positive). She tries several times and each time concludes she is decolorizing too long because both controls have pink cells, one more than the other. What might you suggest she try and why?

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.

what makes acid-fast organisms different from others?

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

what is the mycolic acid?

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

Prepare mixtures of acid-fast and non-acid fast bacteria, stain them with the Kinyoun or Ziehl-Neelsen procedure, and learn to interpret the results accurately.

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

Identify genera that include acid-fast species

Mycobacterium, Nocardia, Cryptosporidium, Isospora

List the reagents utilized in the Kinyoun and Ziehl-Neelsen procedures and identify the purpose of each reagent.

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

Evaluate differences between the Kinyoun and Ziehl-Neelsen methods of acid-fast staining.

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

Identify the composition of the decolorizer.

Explain why acid-fast bacteria are not decolorized

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

Understand why acid-fast bacteria are considered Gram nonreactive

their waxy cell walls repel typical aqueous stains

Explain the role mycolic acids theoretically play in the acid-fast staining reaction

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

Understand the reason for using a drop of serum to prepare the smear for the acid fast stain

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

How does heating the bacterial smear during a ZN stain promote entry of carbolfuchsin into the acid-fast cell wall?

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

Are acid-fast negative cells stained by carbolfuchsin? If so, how can this be a differential stain?

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

Why do you suppose the acid-fast stain is not as widely used as the Gram stain? When is it more useful than the Gram 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

Prepare a mixture of spore forming bacteria and non-spore forming cocci , stain these slides using the Schaeffer-Fulton procedure and learn to interpret the results accurately.

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.

Prepare smears of various spore-forming bacteria and stain these using the Schaeffer-Fulton procedure and learn to interpret the results accurately.

ta-da

Know and understand the purpose of each step in the Schaeffer-Fulton endospore staining procedure. (Be able to identify the decolorizer.)

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

Identify the location of spores within the sporangium. Be able to identify terminal, subterminal and central spores.

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

a state in which there is no metabolic activity

cyptobiotic state

where spores are produced

sporangium

the protein that makes up the tough outer covering of the spore

keratin

responsible for producing and housing the endospore

spore mother cell

Identify three characteristics of spore formation used by microbiologists to help identify bacterial spore formers.

location in cell
shape
size relative to cell

Identify two the bacterial genera that form endospores.

Identify the one known spore forming coccal genera.

Bacillus and Clostridium

Sporosarcina

List two endospore forming genera which also produce antibiotics.

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

why does this exercise call for an older 5-day culture of bacillus?

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

What does a positive result for the spore stain indicate about organisms? what does a negative result for the spore stain indicate about the organism?

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

why is it not necessary to indicate a negative control for this stain procedure?

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

Endospores do not stain easily. Perhaps you have seen them as unstained white objects inside Bacillus species in other staining procedures. If they are visible as unstained objects in other stains, of what use is the endospores stain?

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

a series of controlled transfers down a line of dilution blanks. the series begins with a sample containing an unknown concentration of cells (density) and ends with a very dilute mixture containing only a few cells

serial dilution

agar is solid in tube (leveled) and culture is stabbed into agar

agar pour (deep)

TNTC

too numerous to count

TFTC

too few to count

CP

countable plate

to impress lenticules on a surface or film

lenticulate

cell or group of cells that produces a colony when transferring to a plated media

colony forming unit (CFU)

can be used to determine the number of viable bacteria in a food sample

heterotrophic plate count

growth medium to assess or monitor bacteria growth of a sample

standard methods agar

aka

plate count agar

Differentiate a single cell from a colony-forming unit

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

Understand the process of serial dilution and how one calculates the dilution factor for each dilution made

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

Define a countable plate and understand why plates with fewer and greater numbers of colonies are unreliable

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

should at least produces 30-300, 130 is countable

Understand the basic assumption made concerning the number of bacteria in the inoculum and the number of colonies on a plate

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

Understand the formulas used to determine the concentration of bacteria in the original sample after making plate counts from a dilution series (serial dilution)

x colonies

_________

1x10^{- x} dilution

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

Give reasons a microbiologist (or the health department) might wish to ascertain the number of bacteria per milliliter in a sample of meat.

-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

Know the maximum number of bacteria per gram of meat for the meat to be saleable

must be under 10⁶ bacteria/ gram of meat

Understand the limitations of the procedures used to estimate bacterial numbers

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

You were instructed to add 1.0 mL out of 5.0 mL of an undiluted sample to 99 mL of sterile diluent. Instead,you add all 5.0ml to the 99 mL. What was the intended dilution and what is the actual dilution?

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

a nutrient agar plate labeled 10^-5 mL produced 154 colonies after incubation

what was the cell density in the original sample?

what combination(s) of volumes and dilution factors could have been used to inoculate this plate?

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

1 mL was used to inoculate this plate

a nutrient agar plate labeled 10^-7 mL produced 62 colonies after incubation

what was the cell density in the original sample?

what combination(s) of volumes and dilution factors could have been used to inoculate this plate?

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

1 mL was used to inoculate this plate

why is a standard plate count performed on food products?

to determine number of microbes in 1g of food

if greater than 10⁶ it may be hazardous to consume

Distinguish between transient and resident microbial flora

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

Describe how soap and detergent differ

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

Explain how soap cleans, especially with regard to oily or greasy substances

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.

Understand why soap is not an effective cleansing agent when used with hard water (such as Jacksonville water).

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.

Evaluate the effectiveness of an antiseptic detergent in removing surface organisms

Antiseptic detergents work better than regular soap

Describe the importance of preparing control plates when performing experiments like this one

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.

understand why the single line with zigzag was used to separate the organisms (CFU=s) for this experiment

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

Praise Jesus

yippee!