Microbiology LAB unit 1 test

Describe how to use the two most common types of pipettes?

Blow out pipette- the final few drops of liquid must be emptied in order to deliver correct volume To-deliver pipette- liquid remains in pipette after correct amount is dispensed

What are the tree main types (in terms of physical forms) of microbial culture media?

Broth, semi-solid, and solid

Colony forming units

CFU/mL vs Cells/mL

What is the source of carbon in the chemically defined medium in table 13.1? The source of nitrogen?

Carbon source: Glucose Nitrogen source: Sulfate

How would you determine whether a culture media given to you by the laboratory instructor are sterile before use?

Check media for damages, abnormal smell, and/or growth

Biocide

Chemical control agent- must be determined by effectiveness with respect to the Phenol Coefficient (PC)

Describe the form of some typical bacterial colonies.

Circular- round like a ball Filamentous- fibery Spindle- elongated Irregular Rhizoid- round central shape with branches protruding out

What is a bacterial colony?

Cluster of cells on solid medium visible to naked eye

Why is time an important factor in simple staining?

It allows the dye to create a contrast and stain the bacteria enough that it will not wash off when rinsing the slide

Why is it necessary to use only diluted clusters that contain 100 to 300 cells for a successful spread plate?

It allows to get get single isolated bacterial colonies on a plate

In subculturing, when do you use the inoculating loop?

It transfers the culture from a broth

What is the purpose of subculturing?

Keeps cells/ microorganisms alive by transferring them from culture to new medium to continue to grow and multiply

Why should you use an inoculating needle when making smears from solid media? An inoculating loop from liquid media?

Solid media is more dense over a smaller area, an inoculating needle obtains not too much, a liquid medium has more spread out specimen so the inoculating loop can collect more liquid and enough specimen

Does each discrete colony represent the growth of one cell? Explain. Why can a single colony on a plate be used to start a pure culture?

Yes, when cells are dispersed far enough apart they can multiply into a single pure colony. Then, the pure colony can be extracted and used to make a pure culture.

Differential media

distinguishes between differential groups of bacteria, permits tentative identification based on characteristics and reactions (blood agar medium differentiates between hemolytic and nonhemolytic bacteria)

What are some limitations of a test such as you performed on the evaluation of a disinfectant?

evaporation test only 2 types of bacteria medium deactivated disinfectant time

Selective media

favor growth of particular microorganism and inhibit growth of others (dyes like crystal violet favors gram negative bacteria and inhibits gram postiive)

List some criteria of a good disinfectant.

kill all microorganisms in 10 minutes or less, easy to use, inexpensive, safe for humans and does not damage the surface

Broth media

liquid media -sued to propagate numbers of microorganisms in fermentation studies, determine bacterial motility, and to promote anaerobic growth *TSB- tryptic soy broth

Objective lens

low power- 10x high dry power- 40x oil immersion- 100x *as magnification increases size of lens becomes smaller, admitting less light *light refracts when passing through air and do not enter objective lens- immersion oil prevents loss of light rays

What is the difference between microbicidal and microbiostatic?

microbicidal: to kill microbiostatic: to inhibit

Endospore

-A resistant, dormant structure within a bacteria cell able to protect and survive unfavorable conditions -produced intracellularly - contain DNA, RNA, ribosomes, enzymes, dipicolinic acid, and calcium ions -special stain- Schaeffer-fulton endospore stain -location can be central, terminal, or subterminal ie- clostridium botulinum, clostridium tetani, bacillus anthracis

Simple staining

(following the steps of a bacteria smear slide) 1. cover smear with desired stain and let it sit for proper amount of time 2. rinse with water 3. blot dry

Microbiostatic

(to inhibit) slows the growth of microbes

Microbicidial

(to kill) kills microbes

Acidic dyes

*not used in lab* have a negatively charged chromophore ie- india ink, congo red

Dilution math

- Remember CFU - When plating 1 mL, the dilution remains the same - When plating 0.1 mL, The dilution decreases by a factor of 10

Gram staining

- cell wall yields differential staining *gram positive- thick peptidoglycan layer *gram negative- thin peptidoglycan layer with lipid based outer membrane - gram positive cells contain teichoic acid - alcohol decolorizes gram negative cells only

Three types of inoculations (in this lab)

- inoculate TSA slant, TSA deep, TSB tube from petri plate containing Staphylococcus epidermidis

How is it possible to contaminate a subculture?

- leaving the lid off too long, leaving the lid off test tube - unsterilized instruments, double dipping - breathing directly on specimen, operator contamination

Aseptic technique

- minimizes contamination

What is the function of the iris diaphragm? The substage condenser?

-Iris diaphragm- controls the amount of light that enters the condenser -Substage condenser- focuses the light on a small area above the stage

streak plate technique

-The bacterial mixture is transferred to the edge of an agar plate with an inoculating loop and then streaked out over the surface in a cross hatching form (loop is resterilized after each 'hatch')

Why do microorganisms differ in their response to disinfectants?

-cell wall structure -difference in chemical makeup

brightfield microscope

-dark imagine against brighter background *2 lenses to magnify *stained and unstained cells

Who invented gram staining?

-invented by Hans Christiam Gram in 1884

Differentiate between the resolving power and the magnifying power of a lens. "What is meant by the term parfocal"?

-magnifying- enlarging the image -resolving- differentiating between objecting in the image -parfocal is the ability to change objectives with little to no adjusting and refocusing

What is the phenol coefficient technique?

-microbicidal efficiency of chemical often determined with respect to phenol -standard to compare disinfectant to phenol coeficient

Performing standard plate count technique

-precisely dilute bacterial broth culture -plate known amounts of each dilution -incubate plates -count colonies -determine CFU/mL in original broth

What is a buffer? What is the buffer system used in this exercise?

-resists change to pH, HCl or NaOH is used

Name three basic stains

1. Crystal Violet 2. Carbolfuchsin 3. Methylene Blue

Bacterial smear preparation

1. Place a small drop of water on a slide 2. with the inoculating loop, aseptically add bacterial culture to the drop of water 3. mix the bacteria with the water and spread it out 4. allow to air dry 5. heat-fix the suspension (smear) by quickly passing the slide through a flame two times.

Gram stain procedure

1. Prepare slide: smear, dry, heat fix 2. Primary stain (Crystal violet) 30 seconds, rinse with distilled water 5 seconds 3. Mordant (Gram's iodine) 1 minute, rinse 4. decolorizing agent (Ethanol or acetone) 15-30 seconds, rinse 5. Counterstain (Safranin) 3 minutes, rinse 6. Blot dry w/ bibulous paper 7. view with immersion oil

Describe three ways for sterilizing culture media and supplies.

1. autoclaving- item is exposed to steam at 121*c for 15+ minutes with 15 lb of pressure 2. dry-heat sterilization- (glassware) item is placed in an electric oven set at between 160*c- 170*c for 2+ hours 3. bacteriological filtration- physical removal of bacteria and large microorganisms from solution through filters (without heat)

Asepetic procedure

1. heat loop 5 seconds in bactincinerator 2. cool loop 10 seconds 3. remove caps of tubes (hold in ring and pinky finger) 4. heat fix necks of tubes 5. do transfer 6. heat fix necks of tubes 7. recap 8. heat loop 5 seconds

What is the purpose of the spread plate technique?

1. isolate colonies 2. ability to count them

What is the purpose of heat fixation?

1. kill organisms 2. adhere organisms to slide

Dilution procedure in lab manual

1. pipette 1 mL E.coli culture to 99 mL of sterile water into bottle 1, and shake 2. pipette 1 mL from bottle 1 into bottle 2 3. pipette 1 mL from bottle 2 into an empty petri plate and add an agar pour, swill lightly and let it set, then pipette 0.1 mL from bottle 2 into an empty petri plate and add an agar pour, swill and let it set *first dilution bottle is 1/100 the strength of the original culture 1/100 = 10^-2

Differential staining

1. uses 2 or more stains to create contrast 2. bacteria differ physically and chemically to distinguish types of bacteria

Streak plate mixed culture

1. using 4 quadrant streak method, sterilize loop and place small amount of S. epidermidis in Q1 corner on TSA plate 2. sterilize loop and mix small amount of C. freundii with S. epidermidis 3. proceed with streaking quadrants with cross hatching and sterilizing the loop in between each quadrant 4. place TSA plate in incubator upside down

A disinfectant diluted 1/500 with water kills a bacterium after 10 minutes but not after 5 minutes. a 1/100 dilution of phenol kills the same bacterium after 10 minutes but not after 5 minutes, what is the phenol coefficient of the disinfectant?

500/100= 5 times more effective than phenol

If 5x instead of 10x oculars were used in your microscope with the same objectives, what magnifications would be achieved?

5x X 10x= 50x 5x X 40x= 200x 5x X 100x= 500x

Which of these bacteria are least likely to produce an endospore in their described environment?

A salt-intolerant (nonhalophile) bacterium in freshwater

spread plate technique

A small volume of sample is pipetted onto the surface of the plate A sterile spreading tool or "hockey stick" is used to spread the sample around evenly on the surface to form individual colonies

Why are basic dyes more successful in staining bacteria than acidic dyes?

Bacterial cell walls have a negative charge and basic dyes have an overall positive charged chromophore creating strong ionic interactions

Why is oil necessary when using the 90x to 100x objective?

As magnification increases, resolving power decreases due to light rays being refracted and are lost. Immersion oil catches the light rays and reduces the amount of light refracted, increasing the amount that will be able to be seen

What are 3 bacterial shapes you observed?

Coccus Bacillus Spirilla

How would you define a properly prepared bacterial smear?

Contains evenly spread out bacteria that is properly adhered to the slide through heat fixing

Define culture medium, defined or synthetic medium, and complex or non synthetic medium.

Culture medium- nutrient substance that is used to cultivate microorganisms Defined (synthetic) medium- medium composed of known amounts of pure chemicals Complex (nonsynthetic)medium- medium composes of complex materials rich in vitamins and nutrients (most common are beef, yeast, and peptone extracts)

What is the purpose of simple staining?

Determines the size, shape, and arrangements of bacteria

Colony

Discrete mound of cells on solid medium

Pipette

Instrument to transfer aliquots of culture, prepare serial dilutions, and dispense chemical reagents

How can a streak plate become contaminated?

If it was exposed to air too long, the loop wasn't flamed long enough.

Name the reagent used and state the purpose of each of the following in the Gram stain:

Mordant- Gram iodine, increases the interaction between the dye and the cell wall, so that the cell is able to stain more. Primary Stain- Crystal Violet, stains the bacteria that are gram positive Decolorizer- Used to wash out the crystal violet using 95% ethanol. Counterstain- Safranin, used to stain the bacteria gram negative.

Microbial culture media

Nutrient preparations that are used for culturing microorganisms

In microbiology, what is the most commonly used objective? Explain your answer.

Oil immersion (100X), because it provides the best magnification with the best resolution

Why must young cultures be used when doing a Gram stain?

When gram positive bacteria become too old they stain Gram negative.

A student is working with an unknown bacterium and decides to perform a Gram stain on her organism to start to identify it. After following the procedure, she goes to look at her slide under the microscope, but is unable to see anything. What mistake could the student have made during her process?

She did not heat fix her slide, so her bacteria were removed by the ethanol

What is the difference between a simple and a differential stain?

Simple staining makes the bacteria stand out from their environment, while differential staining distinguishes different types of bacteria

What part of the bacterial cell is most involved with Gram staining, and why?

The cell wall This is because the two types of gram bacteria, gram positive and gram negative bacteria have different peptidoglycan. Gram negative bacteria have cell wall of a thin layer and gram positive have a thick layer. They give different results when stained

Which area of a streak plate will contain the greatest amount of growth? Which area of a streak plate will contain the least amount of growth? Explain.

The first section will have the greatest amount of growth because it the colonies were coming directly from the sample. The other sections contain less due to sterilizing the loop and using a small amount of cultures form the previous section. The 4th section will have the least because it has gone through 2 other sections that contain less and less bacteria

Why are petri plates labeled on the bottom and not the lids?

The lid may contain contamination, the lid can get mixed up with other lids- labeling agar side reduces risks

In the streak-plate technique, how are microorganisms diluted and spread out to form individual colonies?

The plate is separated into 4 sections. Use a loop to inoculate onto one section, sterilize the loop and cool, then, drag colonies from the first section to the second and spread in that secontion, repeat sterilization and dragging into the third and fourth

While performing a Gram staining procedure during lab, a student views their stained slide under the microscope and views two different shaped bacteria that are both stained pink. Knowing that one of these bacteria is Gram-positive and the other is Gram-negative, the student begins thinking about what they could have possibly done wrong. Which of the following scenarios would not have produced this result?

The student did not heat fix their slide, so the color was not retained by the bacteria as well

Why can mannitol salt agar and EMB agar be described as both selective and differential media?

They encourage the growth of both gram negative and gram positive bacteria. Mannitol salt agar is used to isolate staphylococci from clinical and nonclinical samples. EMB agar is used for the detection of E. coli and related bacteria in water supplies and elsewhere.

Why are Petri plates inverted after they cool?

To avoid condensation dripping onto media and cultures and to prevent drying out

What is the purpose of the ethanol in the spread plate technique?

To ensure sterilization of the object being used to spread the bacteria on the plate

Why are culture media sterilized before use?

To get rid of all preexisting bacteria in the media that could affect the experiment and observation

What is the purpose of flaming in the aseptic technique?

To kill any contaminants on the loop/needle that they could of come in contact with in the air or culture

Why is culture medium cooled to about 48° to 50°C before it is poured into Petri plates?

To prevent steam condensation and heat that could damage growth in the plate

True or false: A spread plate is designed to display all of the different microbes present in any sample, and microbes should not be considered to be pure cultures from a spread plate due to the possibility of contamination from other microbes on the plate.

True

Autoclaving sterilization

Used for almost all media Items are sterilized by exposure to 121*C steam, 15 lbs of pressure, for 25 minutes -rapid and dependable method

dry heat sterilization

Used for glassware, pipettes, petri plates Item is put into oven set at 260*C- 170*C for 2+ hours

Which step is the most crucial or most likely to cause poor results in the Gram stain? Why?

Using the decolorizer, species will begin to differ from each other.

Bacterial smear

a thin layer of bacteria placed on a slide for staining

phenol coefficient

calculated by dividing the highest dilution of the antimicrobial of interest by the highest dilution of phenol that has the same characteristics -chemicals with phenol co. greater than 1 are more effective than phenol, less than 1 is less effective than phenol

UV radiation sterilization

can be lethal to microorganisms but only used in few situations (like ceilings of classrooms to disinfect surfaces)

Gram variable

cells of the SAME species staining both colors (often older cultures)

What are some signs of growth in a liquid medium?

cloudy solution and smell

Diaphragm

controls the amount of light entering the condenser

Ethylene oxide gas sterilization

covalently attaches to cell proteins for heat sensitive items, rapidly penetrating packing materials

Condenser

focuses light on small area above stage

filtration sterilization

physically removes bacteria and larger microorganisms from a solution to sterilize without heat

Sterilization

process of all living cells, spores, and acellular entities are either destroyed or removed from object or habitat

Gram staining in lab

smeared staphylococcus epidermidis and citrobacter freundii and gram stain and viewed under microscope

Agar media

solid, and semi-solid solidifying agent NOT a nutrient -used for surface growth to observe colony appearance, culture isolations, storage of cultures, and to observe specific biochemical reactions *TSA- tryptic soy agar

What physical factors can influence the activity of a disinfectant?

temp pH humidity

Inoculating loop/needle

tools used to transfer bacteria from a culture to sterile media, used aseptically