BIOL 2051 LAB MIDTERM

Name six categories of colony morphology properties which can be used to describe the growth of a microorganism

1. whole colony shape

2. margin shape

3. elevation

4. optical properties

5. surface characteristics

6. pigementation

What can influence the colony morpholoy properties of a microorganism?

media used

Describe the growth medium used for experiment one. Would a broth culture work just as well?

TSA (trypticase soy agar) is a solid medium in a petri dish

Using a broth culture would make it difficult to determine colony morphology because the microorganisms are suspended in the liquid. We would be unable to see colony morphology.

Some areas sampled had more microorganisms capable of growing on TSA than other areas. Give a couple of explanations as to why this was observed.

The type and amount of nutrients present in the medium will influence the size, shape, and even color of the colony. The nutrients available in the medium may not have been suitable for growth.

Most of the areas sampled that had a high average number of colony morphologies were high traffic areas that would regularly come into contact with these microbes. The area with the lowest averages are areas that may be cleaned more regularly or do not come into contact with as many microbes as high traffic areas.

Do microorganisms live in all the areas sampled or might some just be transiently there? List the areas sampled where microorganisms are most likely to be residents. Consider the areas where transient microbes were found. How did the microbes get to these areas?

Areas with resident microbes could be mouth/nose, fingers, and hair. These areas can also contain transient microbes transferred onto these areas through daily activities. Areas with transient microbes can also be floor, sink, clothes, air, and dirt.

Where did the microbes come from that landed on the agar surface of the plate left open for 30 minutes?

Airborne particles containing microbes could have landed onto the afar plate and stuck to agar. For ex-ample, sneezing, coughing, breathing, etc. could be common methods of transport for these airborne mi-crobes.

Which of the sites tested are sources of contamination when working with cultures at your lab bench? Suggest a way to prevent contamination from each of these sites.

Airborne particles containing microbes could have landed onto the afar plate and stuck to agar. For ex-ample, sneezing, coughing, breathing, etc. could be common methods of transport for these airborne mi-crobes.

Three descriptions of whole colony shape

1. round

2. irregular

3. rhizoid

Three descriptions of margin shape of colony

1. smooth, entire

2. lobate

3. filamentous

Three descriptions of elevation of colony

1. convex

2. umbonate (button-like)

3. flat

Three descriptions of optical properties of colony

1. opaque

2. semi-translucent

3. translucent

_______ means you can not see through it

opaque

____ means you can see through edges of the colony but not the center

semi-translucent

________ means you can see through it

translucent

Pigmentation means ______

color

Which soap tested was the most effective in reducing the number of bacteria present? the least effective?

most effective: softsoap antibacterial liquid soap

least effective: softsoap NON antibacterial liquid soap

Since most of the normal flora is not harmful, why must it be removed in a surgical scrub?

they could be harmful if introduced into the bloodstream or tissue; to prevent nosocomial or hospital-acquired infections

Why would liquid soap be used in a surgical scrub instead of bar soap?

liquid soap would lessen the probability of cross contamination between the uses of bar soap

Also liquid soap dries more quickly without residue and is more effective

Why could there be more bacteria present after hand washing?

washing skin with soap and water sloughs off the dead outermost layers of the skin and may expose the normal flora

What is meant by the term normal flora?

Microbes that establish themselves on or in the body, but do not produce disease under normal conditions

Why must the inoculating loop be flamed between each quadrant? Flaming is what type of sterilization method?

Flaming the inoculating loop between each quadrant preserves the dilution you're establishing

If you don't flame it, you risk carrying over more bacteria and invalidating the streaking procedure

Flaming is aseptic technique

What is the difference between a mixed culture and a pure culture?

mixed culture=multiple species

pure culture=1 species

What is meant by the term colony? Is a colony a pure culture

A colony is a grouping of genetically identical bacteria from a single cell on an agar plate

An ISOLATED colony is a pure culture

After examining your streak plate, you notice that three colony types are present while the TA says only two organisms were in the mixed broth. What might have caused this?

The third colony could be a transient microflora in the air that infected the plate when you were performing the streak technique

Note: this is technically contamination, but "contamination" is not suitable answer for midterm

Why is it necessary to isolate individual colonies from a mixed broth?

most lab tests for identifying bacteria require pure cultures. clinical specimens usually contain a mix of different species. the medical technologist has to separate the different species before any other tests can be done. she separates them by doing a streak dilution, after the streak dilution plate has incubated, the technologist chooses bacteria from individual colonies. a colony consists of bacteria all descended from the same founder. thus it is a pure culture

Consider the results of your simple stain and the results from your first steak plate. Do these observations agree?

the simple stain was all dyed the same whereas the first streak plate had both gram positive and gram negative bacteria present (done with differential stain so that pink and purple cells present)

Explain why a mixed broth culture may appear pure on the simple stain, but mixed when different colonies were observed on the streak plate.

a simple stain reveals CELLULAR morphologies but covers all the cells the same whereas different colonies could be observed on the streak plate

differentiating between species because although t species nay have the same cellular morphology they could form very different COLONY morpholgies

What types of microorganisms were found in your biofilm?

protozoa and bacteria

How does your biofilm appear different under phase contrast relative to the Gram stain?

there is no movement in the gram stain slide

Is there a correlation between the type of environment and the type of microorganism found in the biofilm? (For example, do you see Cyanobacteria and algae in samples that were exposed to the sun for most of the day?)

Yes, we examined Micrococcus luteus and Protozoa. Micrococcus luteus is normally found in soil, dust, water and air. Protozoa is present in almost all aquatic or moist environments. Most are free-living and eat bacteria, algae or other protozoa.

What is the advantage of using phase-contrast microscopy over brightfield

Living cells can be examined in their natural state without being previously killed, fixed and stained

What do phase contrast and darkfield microscopes have that brightfield scopes do not?

There are plates or filters in the condenser and objectives that increase the contrast between cells and their surroundings without staining to allow for viewing of live cells

Explain the "rotary engine" model for how a bacterial flagellum rotates.

A proton gradient (Na+, K+, and Rb+ can also be used) provides energy to rate the flagellum. The force to spin the motor proteins can be obtained when protons from the periplasm flow back into the cell through Mot proteins, which are located adjacent to the motor.

How do the two flagella of the eukaryotic green algae Chlamydomonas reinhardtii move? Could you see the flagella when viewing the live Chlamydomonas cells?

Pair of flagella at one end of the cell each whipping back and forth to move the cell. Yes, you could faintly see the flagella.

_______ flagella is polar attachment with a single flagellum attached at one end of the cell

monotrichous

___________ flagella is polar; a tuft of flagella attached at 1 or both ends

lophotrichous

___________ flagella is polar; 1 flagella @ each end

amphitrichous

__________ flagellla is non polar attachments; found at multiple locations of the bacterial cell, not just ends

peritrichous

Why does motility test medium have a low agar concentration?

to allow for movement of motile bacteria

Explain the color change that occurs in the motility test medium after inoculation. TTC

TTC, tetrazolium salt, is in the medium. It's an electron acceptor. Oxidized = colorless and soluble, reduced = colorful and insoluble. If the bacteria are motile, then the red color will radiate in all directions away from the original stab line. The entire deep may even turn red. If the bacteria are not motile then you will see a rid line only where you stabbed the deep.

Describe and explain the appearance of a positive and negative motility medium test result

Red radiating multidirectional (very pronounced) is POSITIVE.

Red in only the direction of insertion = NEGATIVE, meaning the microbe didn't move beyond where it was placed.

Beef extract in Nutrient Agar makes NA a __________medium.

complex

________ is composed of digests of chemically undefined substances such as yeast and meat extracts

complex medium

What is the difference between TSA and TSB?

TSA (Tryptic Soy Agar) is a type of solid growth medium, usually containing agar

TSM (Trypic Soy Broth) is a type of liquid growth medium

__________ medium is a nutrient medium designed to favor the growth of certain microbes and inhibit undesirable competitors

selective

________ medium is medium which provides a visible indication of a physiological characteristic of a microorganism

differential

differential or selective medium?

An agar plate that only allows the growth of Gram bacteria

selective

differential or selective medium?

an agar deep that allows you to see if bacteria produces an enzyme

differential

differential or selective medium?

an agar deep that only allows the growth of bacteria capable of using citrate as a carbon source

selective

differential or selective medium?

a flask of brtoh that contains no nitrogen, allowing only the growth of bacteria that can use nitrogen from the air

selective

differential or selective medium?

an agar plate that turns color if the bacteria can produce acidic fermentation products

differential

differential or selective medium?

a tube of broth with a Durham tube to collect gas if it's produced by bacteria growing in the tube

differential

What sealed device is used to sterilize the growth media used in this lab?

autoclave

_______ is a sealed device that allows the entrance of steam under pressure

autoclave

An autoclave heats to _______ C at ______ psi for at least 15 min-1 hour

121

15

When would the following sterilization techniques be used?

dry-heat sterilization

when flaming inoculating loops and inoculating needles

When would the following sterilization techniques be used?

filter sterilization

to physically remove bacteria and other contaminants from heat-sensitive liquids or gases

When would the following sterilization techniques be used?

cold sterilization

to sterilize heat sensitive objects like petri dishes, paper, leather, wood, metal and rubber products

it is also used by NASA to sterilize interplanetary spacecraft

When would the following sterilization techniques be used?

UV radiation sterilization

for exposed surfaces

When would the following sterilization techniques be used?

ionizing radiating sterilization

used in the food industry and for sterilization and decontamination of medical supplies

When would the following sterilization techniques be used?

hot air sterilization

glassware

When would the following sterilization techniques be used?

electromagnetic radiation

microwaves, UV radiation, X-rays, gamma rays, electrons

___________ sterilization kills oxidation effects

dry heat sterilization

A ________ is a device with pores too small for the passage of microorganisms but large enough to allow passage of the liquid or gas

filter

A _______ filter is a touch disc, composed of cellulose acetate or cellulose, which contains a large number of tiny wholes

membrane

in ________ sterilization, the surface of the filter traps particles while allowing the liquid to pass through

filter

________ sterilization uses gas chemo sterilizers

cold

In _____ sterilization ethylene oxide is made use of the plastic petri dish and plastic syringe possible. Ethylene oxide gas is toxic and explosive and is released into a tightly sealed chamber where it circulates for up to 4 hours with the items to be sterilized

cold

_______ sterilization causes damage to the DNA (thymine dimers) leading to the death of exposed organisms.

UV radiation

UV radiation _________ penetrate solid, opaque, light-absorbing surfaces

cannot

___________ sterilization causes ions and other reactive molecules to be produced and these reactive molecules can degrade or alter biopolymers such as DNA and proteins

ionizing radiation sterilization

How does smear preparation for a direct stain differ if the culture is in a broth versus on an agar plate?

When obtaining from a culture in an agar plate, you must place 1 drop of water with the loop on the slide and you mix the cells with the loop in the water on the slide. You do not add water if the culture is in broth.

What instrument is used to obtain culture for a smear from a broth or solid medium?

inoculating loop

The slide warmer does two things in preparing a smear. What are they? Do you do this if preparing a smear for an indirect stain?

Kills the cells, destroying autolytic enzymes and helps the cells to adhere to the slide

No, you do not do this if preparing a smear for an indirect stain because the spear is made in the primary stain and the smear is never rinsed in the staining procedure

If no slide warmer was available, how would you dry and heat fix your smear?

Allow smear to air dry by placing the slide on the bench top for several minute Do not dry the smear over the flame, as this may create an aerosol and cause the cells to rupture

When the smear has air dried, heat fix it by passing the slide over the flame back and forth

The glass heats quickly, so you may want to hold the slide with a clothespin

Do not hold the slide in the flame; excessive heat will damage the cells

Compare and contrast the procedure for a direct stain and an indirect stain.

For direct staining: Following drying and heat fixing, place the prepared smear on a staining rack over the water trough and flood with any one of the available dyes. Leave the stain on the smear for two minutes. Rinse the slide gently with water for a few seconds and blot with bibulous paper. Take care not to wipe the smear off the slide; only blot the excess water from the slide.

For indirect staining: Following air drying of the smear in the primary stain, place the prepared smear on a staining rack over the water trough and flood with acid-alcohol for a couple seconds until the smear turns blue. Drain the excess acidalcohol from the slide. Do not rinse with water. Allow slide to air dry. Do not blot dry.

Look at the genus names of the organisms stained. Do some of them indicate what the cell morphology and cell grouping should be? Which ones? What should the morphology and cell grouping be?

Streptococcus - chained round cells

Staphylococcus - round, random or grape like clusters

Bacillus - rod shaped

Spirosoma - twisted; resemble corkscrews

Vibrio - incomplete spiral

List the cell shapes and give a brief description of each. What are the two most common cell shapes?

Spherical—coccus (pl. cocci)

rod shaped—bacillus (pl. bacilli)

spiral—spirillum (pl. spirilla), spiral with an axial rod—spirochetes

incomplete spiral—vibrio (pl. vibrios)

irregular or variable shape—pleomorphic

The two most common shapes are COCCUS and BACILLUS

Explain why staphylobacilli do not exist.

Because bacilli only divide across their short axis so they will never form a cluster of rods

5 types of coccus

1. single coccus

2. diplococcus (pairs)

3. staphylococcus (random or grapelike clusters)

4. streptococcus (chains)

5. tetrad or cubical packets

3 types of rod-shapes

1. single bacillus

2, streptobacillus (chains)

3. filamentous

What are two genera that commonly form endospores? What is the Gram reaction and morphology of these bacteria?

Bacillus and Clostridium

gram + rods

Are endospores reproductive structures? Why or why not?

No, they are only produced under stressful conditions. An endospore Is a specialized DORMANT structure. Will form within a genetically capable cell when essential nutrients are depleted or when water Is unavailable.

Describe the process of sporulation. When will sporulation occur?

Occurs when a vegetative cell (reproductive) produces endospore (dormant); when nutrients or water becomes depleted and the environmental conditions are harsh.

What component of an endospore protects against harsh chemicals? What component protects the DNA from damage?

The spore coat is responsible for the resistance of the endospore to harsh chemicals; Endospores also contain high levels of calcium complexed with the dipicolinic acid. This complex may play a role in 23 protecting the spore DNA from damage.

What are three common diseases associated with Gram-positive spore forming rods? How does the presence of endospores in these bacteria contribute to the capacity to cause disease?

Botulism, anthrax, and tetanus; endospores allow them to survive during harsh conditions

What is the primary stain, decolorizer, and counterstain used in the staining of endospores?

primary stain: malachite green--decolorizer: water--counterstain: safranin

What chemical is responsible for the acid-fast property of Mycobacterium species?

mycolic acids

What was the appearance of Mycobacterium smegmatis on the agar plate? Why?

compact, wrinkled colonies because of the hydrophobic nature of the cell wall

Why was heat used with the acid fast stain

as the mordant-driving stain into Mycobacterium cells; enhance penetration of the fuchsin

What are the primary stain, decolorizer, and counter stain used when performing an acid-fast stain?

primary stain: carbolfuchsin

decolorizer: acid alcohol

counter stain: methylene blue

What important genus of bacteria is the acid fast stain used to identify?

Mycobacterium

Are acid-fast bacteria Gram (+) or Gram (−)? Explain.

gram positive

cell wall contains peptidoglycan

What are two diseases caused by members of the genus Mycobacterium?

tuberculosis

Hansen's disease

Why is this stain (acid fast stain) so useful for clinical diagnosis?

it can quickly identify the harmful microorganisms

What is the main component of the capsule? What are other components?

Water is the principle component of the capsule; Layers of polysaccharide or polypeptide containing material which surround the cell wall of bacteria

Why was the smear not heat fixed in capsule stain?

because it will destroy the capsule because water is the main component

What are four possible functions of the capsule?

1. desiccation resistance

2. attachment of certain pathogenic microorganisms

3. resist being engulfed by phagocytic cells

4. reserve energy store

Describe the appearance of the capsule-forming bacteria on the surface of the agar. Why might this indicate that it possesses a capsule?

Reddish-pink with white spaces because the cell is red

Why is it important to stain the background with this capsule stain?

the background appears dark purple-red, the actual cells are red to reddish brown while the capsules remain unstained and appear as a clear area surrounding the cell