BMB 305 Pt. 2 Exam 1

purpose of biosafety levels

purpose of biosafety levels

are used to identify the protective measures needed in a laboratory setting to protect workers, the environment, and the public.

Purpose and technique of quadrant streak plate

to obtain isolated colonies from a specimen

Which medium was most difficult for you to transfer from? Which medium was most difficult for you to inoculate? Why? (1.4)

Most people don't like working with slants (solid)

If you got growth on sterile NA and NB slant rubes, why? (1.4)

Possible contamination include not heating the loop to orange-hot, not holding the open tubes on an angle, and/or placing the cap on the table surface during the process.

Did you notice a difference in density (turbidity) of growth in NB tubes inoculated from NB and NA slants? Possible reasons?( 1.4)

Generally, more cells are transferred from growth on a solid medium than from a broth culture. Therefore, broth cultures made from growth on a solid medium will show greater turbidity than those inoculated from a broth culture.

Did you notice a difference in density of growth on NA slants inoculated from NA slants and NB? (1.4)

Generally there will be denser growth on the slant inoculated from the NA slant, because more cells are transferred from solid medium than broth.

Pure culture (1.4)

When a culture ( a medium that contains living microbes) contains a single species.

Broths (1.4)

A medium used to grow microbes when fresh cultures or large numbers of cells are required. Used for ID.

Agar Slant (1.4)

A type of medium used to grow stock cultures that can be refrigerated after incubation and maintained for several weeks.

Plated Media (1.4)

A type of medium used for obtaining isolation of species, differential testing, and quantifying bacterial densities.

Inoculating loops (1.4)

An instrument used to inoculate a medium.

Inoculation (1.4)

To introduce (cells or organisms) into a culture medium.

Using a pencil, draw a quadrant streak (1.5)

Should look like this. You should also rotate a little less than 90 Degrees each streak, and heat the loop so you can get good results. Also, let the loop cool!!

Mixed Culture (1.5)

A microbial culture consisting of two or more species.

What is generally the first step in identifying a microbial organism? (1.5)

Obtaining isolations of Individual colonies. The technique we used in class was the isolation technique- the streak plate. Cells that have been sufficiently isolated will grow into colonies, consisting only of the original cell type.

Colonies (1.5)

Individual microbial cell type. They can also form from a pair, chain, or cluster of cells.

Colony Forming Unit (CFU) (1.5)

A more correct description of the colony origin

Zigzag Inoculation (1.5)

A type of inoculation pattern used to when the sample does not have a high enough cell density.

Quadrant streak technique: (1.5)

• how much space should you try to use?

• describe what each of the quadrants should look like

• describe order

• maximize the space used

• Q1: confluent growth (colonies overlapping), max growth

• Q2: less growth, some separation

• Q3: even less growth

• Q4: isolated colonies

• flame, only go to culture once at the beginning, Q1, flame, Q2, flame, Q3, flame, Q4, flame

Quadrant plate questions: (1.5)

• What if no overlap?

• too much overlap?

• plate with white and yellow colonies is what type of plate?

• no overlap --> no isolation

• too much overlap --> too much confluent growth

• white colonies & yellow colonies --> mixed plate

Confluent Growth (1.5)

When you have confluent growth, the parental bacteria are too close together in space, and so as they reproduce and their progeny reproduce, you see a mass of cells that cover the surface of the agar - and in this case, it isn't possible to separate out which bacteria are the descendants from a single original cell.

Most colonies on streak plates grow from isolated colony-forming units. On rare occasions, however, a colony can be a mixture of two different organisms. If a culture is started from this colony (thinking it's pure), correct identification will be next to impossible because the extra organism could confound the identifying test results. How could you verify the purity of a colony? If you found the colony to be a mixture of organisms, what could you do to purify it? (1.5)

• If you had a mixture of two different organisms, you would have to re-do the quadrant streak

• you could verify the purity of your colony by attempting/conducting a zigzag or quadrant streak and determining if the colonies are identical

• if you do have a mixture, you may also do more quadrant streaks until it's pure

• You could also perform a Gram stain on the isolated culture. If the cells are all the same size, shape, arrangement and gram reaction, then you can be fairly certain the colony is "pure." If you found a mixture of cells, then you should perform a streak plate or a serial dilution pour plate to attempt to isolate a single species of organism. If you are trying for a specific bacterial species such as Staphylococcus aureus or Escherichia coli, you could streak on a selective and differential medium.

Why was the zigzag streak method appropriate to the cell density in the environmental sample? (1.5)

because it was low-density

Difference between colony and CFU 1.5

CFU is theoretical, colony is what you actually see

Light Microscope: 1.Name four lenses & list their magnification

2.What is the magnification of the ocular lens

3. What is the total magnification? 3.1

• scanning: 4x

• low-power: 10x

• high-power/high-dry: 40x

• oil immersion: 100x

• ocular lens magnification = 10x

total magnification

• scanning: 40x

• low-power: 100x

• high-power/high-dry: 400x

• oil-immersion: 1000x

Why aren't the magnifications of BOTH ocular lenses of a binocular microscope used to calculate total magnification? 3.1

• the additional lens does not magnify the image more, instead, both lenses allow both eyes to see the image at the same magnification

What is the total magnification for each lens setting on a microscope with 15x oculars and 4x, 10x, 45x, and 97x objective lenses? 3.1

MT = 15 * 4 = 60x MT = 15 * 10 = 150x MT = 15 * 45 = 675x MT = 15 * 97 = 1455x

Assuming that all other variables remain constant, explain why light of shorter wavelengths will produce a clearer image than light of longer wavelengths? 3.1

Limit of resolution (D) = wavelength / (NA condenser + NA objective)

A small wavelength will allow for a smaller D value. Limit of Resolution is defined as the distance apart two objects must be for the microscope to distinguish them as separate objects. A small D value means better resolution, thus better clarity.

Why is wavelength the main limiting factor on limit of resolution in light microscopy? 3.1

• we cannot see light at a wavelength lower than 380 nm

On a given microscope, the numerical apertures of the condenser and low power objective lens are 1.25 and 0.25 respectively. You are supplied with a filter that selects a wavelength of 520 nm.

A) find D B) will you be able to distinguish two points that are 300 nm apart as being separate or will they blur into one image? 3.1

A)

D = wavelength / (NA c + NA o) D = 520 nm/(1.25 + 0.25) = 346.7 nm

B) No, the two objects must be at least 346.7 nm (D) apart for you to distinguish them as separate objects

On the same microscope as the previous question, the high dry objective lens has a NA = 0.85

A) find D B) Will you be able to distinguish two points that are 300 nm apart as being separate, or will they blur into one image? 3.1

A)

D = wavelength / (NA c + NA ob) D = 520 nm/(1.25 + 0.85) = 247.6 nm

B) Yes, you will be able to distinguish the two points because they are more than 247.6 nm apart

Find D for the oil lens of your microscope. Assume an average wavelength of 500 nm: 3.1

D = wavelength/(NA c + NA ob) D = 500/ (1.25 + 1.25) = 200 nm

Note: you don't have to memorize NA values, will be given BUT know that there is a different NA value for oil immersion

Name parts of the condenser:

What does the filter do?

Which one is the fine adjustment knob?

Which one is the course adjustment knob?

Which one is the mechanical stage adjustment knob?

where is nosepiece?

Where is the condenser?

Describe the relative locations of the iris diaphragm and the filter

3.1

iris diaphragm & lens

the filter is a colored filter, changes color of light used

FA is smaller knob on the side

CA is bigger knob on the side

mechanical stage adjustment knob is hanging down from back corner of stage

nosepiece is above objective lenses; it is a rotating nose piece - rotates the lenses used

• condenser: locate middle of stage, its under there

• iris diaphragm is between stage and filter; filter is below iris diaphragm

What is the formula for calculating total magnification? 3.1

Mt = Mocular * M objective

Define resolution:

Define limit of resolution (D): 3.1

• resolution: resolving power, it is detail (not magnification)

• limit of resolution (D) is the distance apart two objects must be for the microscope to distinguish them as separate objects; is inverse of D; a smaller distance would be a sharper image

the closer they can be at which you can distinguish them (low value of D) --> the better

Formula for D:

What does a smaller D mean?

What is the role of wavelength? 3.1

D = wavelength / (NA condenser + NA objective)

A smaller D means better resolution (think: smaller limit of resolution --> better resolution)

Role of wavelength is color

How can wavelength be manipulated to have a lower D value?

What color has the smallest wavelength? 3.1

select a color with a lower wavelength to have a lower D (limit of resolution) thus better resolution

violet has smallest wavelength

Define Numerical Aperture (NA):

What are the two light capturing lenses in microscope?

What is the purpose of the condenser? 3.1

NA is the measure of a len's ability to capture light

objective lens & condenser are the two light capturing lenses in a microscope

purpose of condenser is not to magnify, but to focus light on specimen

Assume D = 496 nm. What distance apart must two points be in order to be distinguishable? 3.1

More than 496 nm apart to be distinguishable More than D

Why do we have a different NA for oil immersion? 3.1

With use of oil, lens is enabled to capture more light

• oil fills the space between the specimen and lens, so light cannot escape (bend/refract), as a result - more light rays enter lens

Lets say you want to make your resolution better, how could you? 3.1

use a blue-violet filter --> reduces the wavelength

Define working distance:

Describe relationship between objective lens and iris diaphragm

Describe relationship between objective lens and working distance: 3.1

distance between lens and specimen

with scanning, you need minimal iris diaphragm opening (minimal light needed) with oil, you need maximum iris diaphragm opening (most light needed)

• this is because with scanning, you have a high WD -> a lot of space for external light to enter

• with oil, WD is so small, there is barely any space for external light to enter

scanning objective has highest working distance, oil has lowest

Define Parfocal: 3.1

when one lens is in focus, other lenses will also have same focal length and can be rotated into position without further major adjustment

Define visual field: 3.1

the circular area viewed at a given time - visual field

Oil imersion's effect on

• refractions

• D

• NA 3.1

• lowers refractions

• lowers D

• increases NA

What is the point of the wax pencil mark? 3.1

helps you focus on the TOP of the slide

Which objective lens has the smallest visual field? Which one has the highest visual field? 3.1

smallest visual field - OiI largest visual field - scanning

Supergroup Excavata

1. Supergroup containing unicellular organisms

2. Excavated feeding groove on one side of the cell

3. Flagella

4. Four subgroups, parabasalids, diplomonads, kinetoplastids, and euglenozoans

Parabasalids

1. Subgroup of Supergroup Excavata

2. Four anterior flagella

3. Hydrogenosomes that make ATP with H2 as an end product instead of O2

4. Undulating membrane (fin-like extension of the plasma membrane)

5. Picture of Trichomonas vaginalis

Diplomonads

1. Subgroup of Supergroup Excavata

2. Two large nuclei

3. Mitosomes (degenerated mitochondria that lack the electron transport chain)

4. Use cytoplasmic methods to generate ATP

5. The parabasalid, Trichomonas vaginalis is under this group (identify the nucleus and flagella)

Euglenozoans

1. Subgroup of Supergroup Excavata

2. Green, photosynthetic autotrophs

3. Can be heterotrophs as well, so officially they are mixotrophic

4. Generally have two flagella

5. Red eyespot

Kinetoplastids

1. Subgroup of Supergroup Excavata

2. Mass of DNA called a kinetoplast

Supergroup Archaeplastida

1. Supergroup containing organisms believed to be descended from a cell than engulfed a cyanobacterium (primary endosymbiosis)

2. Autotrophic

3. Most have cell walls of cellulose

Chlorophytes

1. Subgroup of Archaeplastida

2. Green algae

3. May be unicellular, filamentous, of colonial

4. Flagella for motility

Charophytes

1. Subgroup of Archaeplastida

2. More closely related to plants than chlorophytes

Supergroup Chromalveolata

1. Supergroup containing organisms believed to be descended from a cell that engulfed a red algae (secondary endosymbiosis, since the red algae already contained chloroplasts), forming a plastid

2. Two subgroups, alveolates and stramenopiles

Alveolates

1. Subgroup of Chromalveolata

2. May be heterotrophic or autotrophic

3. Membrane-bounded sacs beneath the plasma membrane, called alveoli

4. Contains ciliates and apicomplexans

Stramenophiles

1. Subgroup of Chromalveolata

2. Special flagellum with hairs on it

3. Contains diatoms (even though they lack the special flagellum)

Supergroup Unikonta

1. Supergroup containing heterotrophs

2. Includes amoebas, animals, fungi, and others

Amoebas

1. Type of organism in Supergroup Unikonta

2. Two main groups, classical, or free-living, and entamoeba, or parasites

3. Move using pseudopods

Fungi

1. Type of organism in Supergroup Unikonta

2. Nonmotile

3. Cell walls of chitin

4. Absorptive heterotrophs, that secrete enzymes then absorb the nutrients

5. May be unicellular or filamentous

Undulating Membrane

A sinuous extension of the cytoplasmic membrane performing a vigorous, wavelike, and reversible movements

Simple Stains

1. Contain a solvent and a colored molecule, or chromogen

2. The chromogen contains the chromophore, or portion that gives it its color, and the autochrome, which is charged

3. The charged portion allows the dye to interact with the cell

4. In this type of stain, the autochrome is positively charged (basic, so picks up proton), and interacts with the negative charges on bacterial cells

5. Applied to bacteria that have been heat-fixed to kill the bacteria and make them stick to the slide, but the process may distort the cells and cause them to shrink

6. Bacteria show up as colored against a light background

What is the consequence of leaving a stain on the bacterial smear too long (overstaining?)

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

What is the consequence of not leaving a stain on the bacterial smear long enough (understaining?)

Cell will be clear, difficult to see

Basic Stains

methylene blue, crystal violet, safranin

Steps for simple stain

1. add drop of water to slide

2. add bacteria to water on slide and smear

3. heat fix (put over flame 3 times, 10 secs in between)

4. add stain for 1 min

5. rinse

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.

Negative Staining

1. In this stain, the chromogen has a negative charge (acidic, so loses proton)

2. As a result, it is repelled by the negative charges on the bacteria

3. Bacteria show up as light against a dark background

4. Bacteria are not heat fixed, so shrinkage is minimal, and is helpful when it is important to be able to determine the size of the cells

Negative stains

negrosin, eosin

Negative Staining Steps

1. add drop of negrosin to slide

2. add bacteria to negrosin and smear

3. drag second slide over stain

4. air dry and observe

why doesn't a negative stain colorize cells?

The negative charge of the stain is repelled by the negative charge of the cell wall.

Eosin is red stain and methylene is blue. what should the reult be of staing bacterial smear with mixutre of methylene blue and eosin?

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.

Compare the diameter of M. luteus cells as measured using a basic stain and an acidic stain what might account for any difference?

Basic (simple stain) heat fixing--shrinks cells/smaller negative--cells are their actual size HOWEVER--there is very little difference!!

Gram Stain

1. Differential stain that tells the difference between two types of bacteria, in this case Gram negative and Gram positive

2. Cells are stained first with crystal violet, then with iodine, which helps enhance the crystal violet

3. Afterwards the cells are decolorized with alcohol solution (the thick cell wall of Gram positive bacteria is not penetrated by the alcohol, so the dye is not washed out, while the dye is washed out of the Gram negative bacteria because the alcohol extracts the cell wall lipids and makes it more porous)

4. The cells that were decolorized are retained with safranin

5. Issues include over decolorizing, which makes Gram positive cells dye red

6. Issues also include under decolorizing, which makes Gram negative cells dye purple

7. Older samples may also not stain as well

cell wall types

Gram Positive: thick layer of peptidoglycan. Techoic acid strands. Lipotechoid acid. Only a cell wall.

Gram Negative: outer layer of phosopholipids inner thin layer of paptidoglycan. Outer membrane, inner membrane, and periplasm. LPS on the outside.

Gram Staining Steps

1. Primary stain: crystal violet (basic stain), 1 min rinse

2. mordant: gram iodine 1 min

3. decolorizer: alcohol or acetone

4. counterstaining: safranin (pink)

Mordant

substance that enhances the ability of stain to adhere to cell wall

after decolorizer...

Gram pos- stained blue gram negative- clear -easily removes phospholipid layer

after counter staining....

Gram positive: appears blue

gram negative: appears pink

False results occurs from

1. overdecolorizing (gram + can appear red)

2. underdecolorizing (gram - appear purple)

3. too much material used

4. overcooked

5. old culture (bacteria start to lose cell wall)

In Lab we used...

used escherichia coli- gram neg rod, appeared pink

used micrococcus luteus- gram positive cocci, appear purple

Mistakes in Gram Staining

• Failure to apply iodine--everything will appear gram negative

• Failure to apply the decolorizer--everything will appear gram positive

• Failure to apply the safranin--gram (+) will be purple, gram (-) will be colorless

• Reversal of crystal violet and safranin--wouldn't be able to read and would have to toss it.

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

• Gram (+) can hold on to the crystal violet although it's decolorized. Gram negative bonds to safranin after phospholipid layer is removed

Acid Fast Staining

1. Stain that takes advantage of mycolic acids in the cell walls of some organisms

2. Mycolic acid gives the cells greater affinity for the primary stain and resistant to decolorizing

3. Because the mycolic acid is waxy, a lipid-soluble stain must be used

4. Two methods of staining: the Ziehl-Neelsen method and the Kinyoun method

Ziehl-Neelsen

1. Carbolfuchsin is used as the primary stain

2. Cells are steam-heated before staining to melt the wax and allow the stain to penetrate more easily

3. Acid alcohol is used to decolorize (acid-fast cells are not decolorized)

4. A counterstain is applied, like methylene blue (shows non acid-fast cells)

Kinyoun Method

1. Uses more concentrated and lipid-soluble carbolfuchsin as the primary stain

2. Doesn't use heat, so is a bit less sensitive than the Ziehl-Neelsen method

3. Acid alcohol is used to decolorize

4. A counterstain is applied

Basic Steps for Emulsion Smear

1. Prepare Bacterial Smear Emulsion

• Drop water

• Aeseptically Add bacteria

• Air dry smear

• Pass the smear over outercone flame

• cool the slide

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

Heating melts the mycolic acid and allows the stain to penetrate the cell walls.

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

It uses acid alcohol as a decolorizing agent which extracts the carbolfuchsin from the nonacid-fast cells while ineffective on the acid-fast positive cells. The nonacid-cells are then counterstained with brilliant green to show the difference.

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

Capsule Stain

1. Stains around and inside the cells, so the capsule, which resists stains, appears as a white halo between the cells and the background

2. An acidic stain is applied to stain the background

3. A basic stain is applied to stain the cells

4. Instead of heat-fixing, which can result in artificial halos when the cells shrink, the bacteria may be emulsified in a serum to help them stick to the slide

-differential stain (bacteria with or withou capsules) -reveal the presence of the bacterial capsule -both gram-positive and gram-negative, may be surrounded by an outer polysaccharide-containing layer termed the glycocalyx

Glycocalyx

2 types: capsule: this layer is tightly bound and remains attached to cells slime layers : More loosely bound layers, more easily removed

-Glycolax can not be easily stain but can be penetrated by basic stain to stain bacteria within -Allows bacteria to stick to surfaces and each other -Determines of virulent (pathogenic) bacteria is

def. virulence- degree of pathogenicity, severeity of disease

Capsule

Can be composed of glycoproteins, mucoid polysaccharides, or polypeptides -protective structures, protect cells from phagocytosis -some bacteria always have a capsule ex. Klebsiella pneumonia (can be used as a positive control for capsule staining)

CapsuleStaining Uses 2 Staining Techniques

1. negative stain (acidic): congo red, stains background

2. simple stain (basic +): maneval stain, stains bacteria

no heat fixing: heat shrink cells which can leave a white halo which might look like a capsule

Basic Capsule Staining Steps

1. mix bacteria into smear onto slide

2. add congo red

3. drag another slide across and allow to dry

4. add maneval stain for 1 min

5. rinse

Results: the capsule is revealed as a clear halo between the colored background and the stained cell.

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.

Endospore

endospores serve as a protective structure for survival of the organisms in response to unfavoravle conditions -produced within vegetative cell -can be seen within the vegetative cell or outside of cell (free spore) -coating made of keratin (heat is used to penetrate) -increases virulence

only rod bacteria produce endospores -not all rods produce endospores -two genus that do: Bacilli, Clostridia