Lab Practical #1

Fungal colonies

- type of eukaryotic microbe - tend to be larger compared to prokaryotic microbes - irregular: in their edges and filamentous - texture - major environment: dirt (ground or dust) - color: whitish

Bacteria colony morphology

- type of prokaryotic cell form/shape: circular - round shape margin/edges: entire - smooth with no irregularities elevation: convex - water drop shape (most common bacterial elevation) texture: shiny/smooth - mucus consistency pigment production: yellow/white/beige - Bacillus can differ in their elevation and texture

What type of microbial colonies are found on this plate? a) both fungal and bacterial colonies b) only fungal colonies c) only bacterial colonies d) cannot determine

answer: b) only fungal colonies

Organism C: 4 degrees C ++ 25 degrees C ++ 37 degrees C 55 degrees C Using the above data from a Temperature Experiment, answer the following question. Organism C is best classified as a __________________________. a) Thermophile b) Psychrophile c) Mesophile d) Cannot determine

answer: b) psychrophile

Is this a successful streak plate? a) no, there are no isolated colonies b) yes c) no, there is a contaminant

answer: c) no, there is a contaminant

Be able to evaluate plates from the lab

- measure the diameter of the zone of inhibition with a ruler - 1 cm = 10 mm - can take the average of the zone of inhibition - measure both ways and divide by 2 - if the zones of inhibition are too large, you won't be able to see the complete diameter - measure one radius and multiply by 2 - kirby-bauer assay: need to use a chart to determine if the bacteria is resistant, intermediate or susceptible to the antibiotic

Selective Treatment: Alcohol (chemical treatment)

- most important step in the procedure because it allows one specific structure (gram positive - more layers of peptidoglycan) to retain the primary stain color, while other structures are decolorized (gram negative - only one layer of peptidoglycan) at the completion of the selective treatment - alcohol can break down phospholipids and peptidoglycan, making holes in the cell wall and washing out the complex formed between crystal violet and iodine - gram negative will be clear, since the complex was washed out by the selective treatment and gram positive will still have the purple complex

Obligate Anaerobes

- must be without oxygen to grow - does not have the protective enzymes - uses anaerobic respiration - will see growth at the bottom

Microaerophiles

- needs low levels of oxygen to grow - has little protective enzymes - uses aerobic respiration

Evaluate the success of different gram stains

- no contamination present: one shape only - all of the cells must either be stained pink or purple - avoid false gram negatives (pink) by ensuring the alcohol isn't on long enough to over decolorize the gram positive cells - avoid false gram positives (purple) by applying enough alcohol to weaken the gram negative cell walls - use cultures that are 24-48 hours old - avoid false gram negatives by making sure the iodine step is done

Explain the different oxygen requirements for bacteria

- obligate aerobes - obligate anaerobes - facultative anaerobes - aerotolerant anaerobes - microaerophiles

Psychrotrophs

- optimal temperature: 22 degrees Celsius (room temperature) - temperature range: 0-30 degrees Celsius

Mesophiles

- optimal temperature: 37 degrees Celsius (body temperature) - temperature range: 10-50 degrees Celsius

Thermophiles

- optimal temperature: 65 degrees Celsius - temperature range: 40-70 degrees Celsius

Hyperthermophiles

- optimal temperature: under 100 degrees Celsius - temperature range: 70-110 degrees Celsius

Describe the different temperature requirements for bacteria

- psychrophiles - psychrotrophs - thermophiles - hyperthermophiles - mesophiles

Obligate Aerobes

- requires oxygen to grow - has the protective enzymes - uses aerobic respiration - will see growth at the top

B. subtilis

- shape/form: irregular - non-round shape - margin/edges: undulate - wavy and irregular - elevation: flat - texture: dry/wrinkled - pigment production: off-white/beige

What were the results for the E. coli gram stain?

- shape: bacillus - arrangement: single - color: pink - gram reaction: gram negative

What were the results for the S. epidermidis gram stain?

- shape: cocci - arrangement: staphylo - color: purple - gram reaction: gram positive

Characteristics of colonies

- size, color, texture - these are determined genetically - help with identification of unknowns - any difference seen in morphology indicates a different species

Be able to calculate the total magnification

- total magnification: scanning - 4x multiplied by 10 = 40x - total magnification: 10x multiplied by 10 = 100x - total magnification: high and dry - 40x multiplied by 10 = 400x - total magnification: oil lens - 100x multiplied by 10 = 1000x

Mold colony morphology

- type of eukaryotic cell that is a multicellular fungi form/shape: filamentous - hair-like extensions (mold hyphae) margin/edges: filiform - root-like extensions (mold hyphae) elevation: raised texture: wooly/hairy/moist pigment production: colorless - spores give off color - blue-green

Yeast colony morphology

- type of eukaryotic cell that is a single-celled fungi form/shape: circular - round shape margin/edges: entire - smooth with no irregularities elevation: convex - water drop shape texture: shiny/smooth pigment production: creamy off-white - requires cell visualization under a microscope

Bacterial colonies

- type of prokaryotic microbe - tend to be smaller compared to eukaryotic microbes - circular: little round dots - mucoid: shiny/slimy like mucus - major environment: microbiota or flora (human body) - color: whitish or yellowish

Potential antibacterial chemicals are tested on Blood Agar to determine if they can be used on human tissue. Explain what you would see on the Blood Agar that would indicate the chemical would not be safe to use on human tissue.

If a chemical wasn't safe to use on human tissue then you'd see areas on the blood agar where the red blood cells have begun to lyse, creating a clear and transparent area because the red blood cells within the agar are being destroyed by the chemical. If a chemical has the ability to destroy the red blood cells, then it'd be caustic to human tissue.

You are preparing to perform a Gram stain on two samples, E. coli and S. epidermidis. What results would you see for the E. coli and the S. epidermidis, if: You forget to add the safranin.

E. coli (gram negative): clear bacilli S. epidermidis (gram positive): purple cocci - safranin is what stains E. coli pink

You are preparing to perform a Gram stain on two samples, E. coli and S. epidermidis. What results would you see for the E. coli and the S. epidermidis, if: You forget to add the iodine.

E. coli (gram negative): pink bacilli S. epidermidis (gram positive): pink cocci - S. epidermidis won't have that complex and alcohol can easily wash out the crystal violet, causing them to be clear and safranin will stain them pink

You are preparing to perform a Gram stain on two samples, E. coli and S. epidermidis. What results would you see for the E. coli and the S. epidermidis, if: You forget to add the crystal violet.

E. coli (gram negative): pink bacilli S. epidermidis (gram positive): pink cocci - the safranin would stain them both pink since there was no primary stain added

You perform a Gram stain on two samples, E. coli and S. epidermidis that have been in the incubator for one week. What results would you see for the E. coli and the S. epidermidis?

E. coli (gram negative): pink bacilli S. epidermidis (gram positive): purple and pink cocci

You are preparing to perform a Gram stain on two samples, E. coli and S. epidermidis. What results would you see for the E. coli and the S. epidermidis, if: You forget to add the alcohol.

E. coli (gram negative): purple bacilli S. epidermidis (gram positive): purple cocci - there's no alcohol to disrupt the phospholipids or peptidoglycan and cause the crystal violet to leave the cell wall

This tube shows a(n) _____________________ pattern of growth. a) aerotolerant anaerobic b) obligate anaerobic c) microaerophilic d) obligate aerobic e) facultative anaerobic

answer: a) aerotolerant anaerobic

If a bacteria is a facultative anaerobe then you would expect to see _________________. (Pick all that apply.) a) Growth on the plate from the incubator. b) No growth on the plate from the gas-pack jar.. c) No growth on the plate from the incubator. d) Growth on the plate from the gas-pack jar.

answer: a) and d)

If a bacteria is a obligate aerobe then you would expect to see _________________. (Pick all that apply.) a) Growth on the plate from the incubator. b) No growth on the plate from the incubator. c) Growth on the plate from the gas-pack jar. d) No growth on the plate from the gas-pack jar..

answer: a) and d)

The most likely source (surface that was swabbed) for this plate is _______________. a) bottom of a shoe b) inside a person's mouth c) around a person's fingernail

answer: a) bottom of a shoe

Which of the following can be used to control the amount of light passing through the lenses? (select all that apply) a) condenser b) ocular lenses c) iris diaphragm lever d) fine adjustment knob e) coarse adjustment knob f) light source adjustment knob

answer: a) condenser - located underneath the stage, can move up/down, f) light source adjustment knob, c) iris diaphragm lever - located underneath the stage

Can you determine if the bacteria in your smear can exhibit phototaxis? a) no, because the heat fixing killed them b) no, because the stain would slow them down c) yes, if oil immersion is used d) yes, if the light is not too bright

answer: a) no, because the heat fixing killed them

Is the one on the left a successful streak plate? a) no, there are no isolated colonies b) yes c) no, there is a contaminant

answer: a) no, there are no isolated colonies

What type of microbial colonies are found on this plate? a) only bacterial colonies b) both bacterial and fungal colonies c) cannot determine d) only fungal colonies

answer: a) only bacterial colonies

Which disinfectants affect proteins? (select all that apply) a) Spic and Span b) None of the disinfectants c) Iodine d) Hydrogen Peroxide e) Hand Sanitizer

answer: a) spic and span and e) hand sanitizer

Organism A: 4 degrees C 25 degrees C 37 degrees C 55 degrees C ++ Using the above data from a Temperature Experiment, answer the following question. Organism A is best classified as a __________________________. a) Thermophile b) Psychrophile c) Mesophile d) Cannot determine

answer: a) thermophile

Is this a successful streak plate? a) yes b) no, there is a contaminant c) no, there are no isolated colonies

answer: a) yes

Is it possible for an obligate anaerobe to cause disease in a human? (Pick all that apply.) a) Yes, because there are some areas of the human body where there is no free oxygen. b) Yes, because obligate anaerobes can survive in areas of low oxygen. c) No, because body temperature is too warm for them to replicate. d) No, because there is oxygen in all parts of our bodies.

answer: a) yes, because there are some areas of the human body where there is no free oxygen

Which of the following is true about lowest objective? (select all that apply) a) it has the largest field of view b) it is used to initially locate the specimen c) it can be used for oil immersion d) when using it the object will be magnified by 100 times e) you can use the coarse adjustment knob to adjust the focus

answer: a), b), e)

What type of microbial colonies are found on this plate? a) cannot determine b) both bacterial and fungal colonies c) only fungal colonies d) only bacterial colonies

answer: b) both bacterial and fungal colonies

The Gram stain is used to determine what type of _________ the bacterial cells have. a) arrangement b) cell wall structure c) membrane structure d) shape

answer: b) cell wall structure

How could you determine the measurement of the zone of inhibition associated with the red disk? a) Measure the visible circumference. b) Measure the radius and multiply by two. c) Measure the radius and add two d) You cannot measure the zone of inhibition.

answer: b) measure the radius and multiply by two

This tube shows a(n) _____________________ pattern of growth. a) aerotolerant anaerobic b) obligate anaerobic c) microaerophilic d) obligate aerobic e) facultative anaerobic

answer: b) obligate anaerobic

Is this a successful streak plate? a) no, there are no isolated colonies b) yes c) no, there is a contaminant

answer: b) yes

If an organism grows at 45 degrees C in reducing media, then which of the following could be true? (Pick all that apply.) a) It could be an aerotolerant anaerobe. b) It could be an obligate anaerobe. c) It could be a psychrophile. d) It could be an obligate aerobe. e) It could be a thermophile f) It could be a facultative anaerobe. g) It could be a mesophile.

answer: b), e), f) and g)

This is a plate of Serratia marcescens from the Temperature Experiment. At which temperature was it incubated? a) 55 Degrees C b) Cannot determine c) 37 Degrees C d) 4 Degrees C e) 25 Degrees C

answer: c) 37 degrees celsius

The most likely source (surface that was swabbed) for this plate is _______________. a) surface of a leaf b) bottom of a shoe c) a person's scalp

answer: c) a person's scalp

If a bacteria is a aerotolerant anaerobe then you would expect to see _________________. (Pick all that apply.) a) No growth on the plate from the gas-pack jar.. b) No growth on the plate from the incubator. c) Growth on the plate from the gas-pack jar. d) Growth on the plate from the incubator.

answer: c) and d)

If a bacteria is a obligate anaerobe then you would expect to see _________________. (Pick all that apply.) a) Growth on the plate from the incubator. b) No growth on the plate from the gas-pack jar.. c) Growth on the plate from the gas-pack jar. d) No growth on the plate from the incubator.

answer: c) and d)

Which disinfectant affects cell membranes? (select all that apply) a) Hydrogen Peroxide b) None of the disinfectants c) Hand Sanitizer d) Iodine e) Spic and Span

answer: c) hand sanitizer and d) iodine

Organism B: 4 degrees C 25 degrees C ++ 37 degrees C ++ 55 degrees C Using the above data from a Temperature Experiment, answer the following question. Organism B is best classified as a __________________________. a) Thermophile b) Psychrophile c) Mesophile d) Cannot determine

answer: c) mesophile

Would you use antibiotic G to treat an infection by this bacteria? a) No, because the zone is too small. b) Yes, because there is a zone of inhibition. c) No, because there are resistant bacteria. d) Don't know, because need the table to determine if the bacteria is susceptible, intermediate or resistant.

answer: c) no, because there are resistant bacteria

Which disinfectants affect DNA? (select all that apply) a) Spic and Span b) None of the disinfectants c) Iodine d) Hydrogen Peroxide e) Hand Sanitizer

answer: d) hydrogen peroxide

This tube shows a(n) _____________________ pattern of growth. a) aerotolerant anaerobic b) obligate anaerobic c) microaerophilic d) obligate aerobic e) facultative anaerobic

answer: d) obligate aerobic

What is the purpose of adding water to a smear created from solid media? a) to keep the cells hydrated b) to help the cells stick to the slide c) to allow the stain to adhere to the cells d) to spread out the cells on the slide

answer: d) to spread out the cells on the slide

This tube shows a(n) _____________________ pattern of growth. a) aerotolerant anaerobic b) obligate anaerobic c) microaerophilic d) obligate aerobic e) facultative anaerobic

answer: e) facultative anaerobic

This is an example of

blood cells

Dry/wrinkled, white in color, large and irregular

colonies of B. subtilis (bacteria)

Shiny/mucoid, white/beige in color, small and round

colonies of E. coli (bacteria)

Shiny/mucoid, yellow in color, small and circular

colonies of M. luteus (bacteria)

Shiny/mucoid, white in color, small and round

colonies of S. epidermidis (bacteria)

Mordant: Iodine

enhances/intensifies the primary stain - forms a complex with crystal violet and helps trap the stain within the peptidoglycan (gram positive cell walls have more peptidoglycan and can firmly trap the complex more than the gram negative cell walls can) - both gram positive and gram negative will be a deep purple shade

Lab 2 Streak Plate: Describe the purpose of this lab

purpose: 1) observe individual colonies - mass of microbes that is observable to the naked eye and is derived from a single cell that has been allowed to undergo cell division 2) identify the colony morphology (characteristics) 3) correctly prepare a streak plate

Lab 1 Ubiquity of Microbes: Describe the purpose of this lab

purpose: 1) practice basic microbial culture techniques 2) Identify general types of colonies (prokaryotic and eukaryotic) 3) test the "ubiquity" of microbes

Lab 8 Oxygen Requirements: Describe the purpose of this lab

purpose: 1) understand the different oxygen requirements and classification 2) be able to analyse results to determine the requirements for bacteria

Lab 8 Temperature Requirements: Describe the purpose of this lab

purpose: 1) understand the different temperature requirements and classification 2) be able to analyse results to determine the requirements for bacteria

Lab 9 Disk-Diffusion and Kirby-Bauer: Describe the purpose of this lab

purpose: 1) understand the process of a disk diffusion assay - used to determine the effectiveness of different antimicrobials 2) be able to interpret disk diffusion results 3) be able to interpret Kirby-Bauer results

Lab 5 Gram Stains: Describe the purpose of this lab

purpose: 1) understand the purpose of differential stains (gram stains - cell wall structure) 2) understand the process of a gram stain 3) correctly perform a gram stain

Explain the purpose of thioglycollate tubes and interpret their growth patterns

purpose: acts as a reducing media to chemically reduce free oxygen and inhibit its diffusion - exposes microbes to different concentrations of oxygen in a gradient (closest to the top - aerobic and closest to the bottom -anaerobic) - has an indicator that turns pink when oxygen is present - used to grow facultative anaerobes and obligate anaerobes

Describe the purpose of the Blood agar and Mueller-Hinton media

purpose: blood agar - used to determine if antibacterial chemicals can be used on human tissue - used to grow fastidious bacteria - used to differentiate bacteria based off their hemolytic capabilities purpose: Mueller-Hinton - all purpose media that contain starch: starch will absorb toxins released from bacteria, which will prevent their interference with the antibiotics - slightly less dense than other all-purpose media, which allows better diffusion of the antibiotics and chemical (results in truer zone of inhibition)

Explain the purpose of oil immersion

reduce the refraction of light

What arrangement is this?

sarcinae

What shape is this?

single cocci

What shape is this?

spirillum

What shape is this?

spirochete

What would happen if you placed the slide upside down (sample closer to the stage)?

the image would remain blurry and you could not focus on it

How do you correctly perform a streak plate

tips and tricks: 1) be gentle rubbing the loop on the agar 2) just touch the loop to the bacteria - don't want to grab too much (want to isolate) 3) use a zig zag motion 4) sterilize the loop in between streaks 5) let the loop cool for 30 seconds to avoid killing the bacteria 6) use a sharpie to label the plate and draw a "T" to guide your streaks

What shape is this?

vibrio

This is an example of

Spirochetes

This is an example of

Streptobacilli (chain)

Describe how and when to clean the microscope

- how: use optical lens paper (tissue paper) and lens cleaner - when: before using it, in between slides, and after you use it

Your lab partner makes a smear of a pure culture of E. coli and observes a mixture of cocci and bacilli. Is this possible? If not, what has happened and how can she resolve the issue?

- No, this is not possible, because the shape that is specific to E. coli is bacilli. If there was a mixture of both bacilli and cocci then this means that before making the smear, my lab partner must've been touching the center of the slide, instead of the edges, with her fingers which led to contamination. For example, the bacteria that is found on human skin is Staphylococcus epidermidis and this bacteria has a cocci shape, which would explain why she observed a mixture of bacilli and cocci. - Another thing that could've happened is that she made a smear before that, using bacteria that had a cocci shape and failed to sterilize the loop before making a new smear, which can also lead to contamination. My lab partner can resolve the issue by preparing a new smear and this time, ensuring that she makes the smear using aseptic techniques, such as holding the edge of the slide with a clothespin and making sure she sterilizes the inoculating loop by putting it in the flame of the bunsen burner until it glows orangey-red to prevent further contamination.

Counterstain: Safranin

- a simple basic stain that is a different color from the primary stain, which is used to stain any cellular components that do not have the primary stain (gram negative) allowing contrast between the different cellular components - turns gram negative cells reddish/pink, while gram positive cells remain purple

Primary stain: Gram's crystal violet

- a simple basic stain that penetrates and diffuses through the cell wall and is used to color the cells or a specific component of interest - turns both gram negative and gram positive cells purple

If the same chemicals were tested on the same type of bacteria on a Mueller-Hinton plate, you would expect: a) Same size zones of inhibition. b) Larger zones of inhibition. c) Smaller zones of inhibition. d) Cannot determine what the size of the zones of inhibition would be.

- answer: b) larger zones of inhibition

What shape and arrangement did you observe for E. coli in lab?

- arrangement: single - shape: bacillus

What shape and arrangement did you observe for S. epidermidis in lab?

- arrangement: staphylo (clusters like grapes) - shape: coccous

Facultative Anaerobes

- can grow in areas with or without oxygen - has the protective enzymes - uses oxygen to its benefit - will have denser growth at the top and less dense growth towards the bottom - uses aerobic and anaerobic respiration

Aerotolerant Anaerobes

- can grow in the presence of oxygen, but doesn't use oxygen to metabolize - has protective enzymes - uses anaerobic respiration - growth occurs evenly

Psychrophiles

- cold loving bacteria - optimal temperature: 10 degrees Celsius - temperature range: -10-20 degrees Celsius

Describe the aseptic techniques used in performing the lab

- disinfecting bench - washing hands - using a clothespin to hold the slide - using a bunsen burner to sterilize the loop - flaming the mouth of the culture tube - air drying the slide and not blowing on it or waving it in the air

Colony morphology

- form: the whole colony shape when looking overhead - punctiform: very small colonies, circular: round, irregular: non-round, rhizoid: tree branch (mold hyphae) - elevation: look at the colony from the side and see how it rises above the agar - flat: very close to the agar, raised: way above the agar, convex: curved, pulvinate: high in the middle, umbonate: high center - margin: looking at the outside outline of the colony - entire: even and smooth, undulate: wavy, filamentous: hairy, curled: different waves, lobate: lobes. erose: serrated - texture: surface appearance and consistency - dull, shiny, mucoid, rough and wrinkled - pigment production: colored products produced by the microbe shades of white: pure white, off-white (yeast), cream, beige, shades of yellow: gold, shades of red: crimson, pink, orange, shades of green: blue-green (mold)

Match the disinfectant to the correct active ingredient. 1) Spic and Span a) ammonium compound 2) Hand Sanitizer b) halogen 3) Iodine c) peroxygens 4) Hydrogen Peroxide d) alcohol

1) - a) 2) - d) 3) - b) 4) - c)

Correctly label the microscope

1) ocular lenses 2) objective lenses 3) stage clips 7) light source 8) base 12) nosepiece 13) arm 14) coarse adjustment knob 17) mechanical stage adjustment knob 18) light source adjustment knob 19) on/off switch

Describe the different reagents, the order they are used and their purpose

1) primary stain: gram's crystal violet 2) mordant: iodine 3) selective treatment: alcohol 4) counterstain: safranin

Another student in the class performed the procedure for this experiment perfectly, yet 48 hours after incubation there were no discernible colonies on the plate. Give two different reasons to explain these results. (Mistakes in procedure or equipment are not valid answers.)

1. If the student chose an area, such as the bottom of a shoe, or any other area where fungi may thrive, then the fungi may take longer to grow than 48 hours, therefore, the colonies are probably there, but are just too small to be seen with the naked eye. 2. Also, eukaryotic microbes, such as fungi replicate via cell division, which involves more structures and processes than prokaryotic binary division, therefore, there is a greater chance for mutations to occur, thus disrupting cell division and causing the cell to die.

Describe two different situations when it is important to be able to identify a specific microbe.

1. It's important to be able to identify a specific microbe because eukaryotic microbes and prokaryotic microbes can cause different types of infections, for example, prokaryotes cause bacterial infections, and in order to find a medication that is selectively toxic, meaning it's able to kill the prokaryotic cells without harming the the eukaryotic human cells, then you need to be able to identify the microbe and understand the differences between eukaryotes and prokaryotes 2. It's important to be able to identify a specific microbe because if a cheesemaker wants to make a certain type of cheese, then a specific type of bacteria will need to be identified to meet the cheese characteristics wanted by the cheesemaker

Prokaryotic cells are usually viewed with a total magnification of ___________ and eukaryotic cells are normally viewed with a total magnification of __________.

1000x, 400x

In the Kirby-Bauer experiment, _________A_______ exhibited the largest zones of inhibition and _______B________ exhibited the smallest zones of inhibition overall.

A: Staphylococcus aureus B: Pseudomonas aeringrosa

Explain how an aerotolerant anaerobe differs from a facultative anaerobe.

Aerotolerant anaerobes use anaerobic respiration as their metabolic pathway, but can grow in the presence of oxygen, however, they do not use oxygen to produce ATP. Facultative anaerobes can either use aerobic respiration or fermentation as their metabolic pathway. Therefore, they can grow without oxygen or in the presence of oxygen and use it to their advantage to produce more ATP per glucose molecule. In thioglycollate media, aerotolerant anaerobes will have more even growth throughout the media, whereas facultative anaerobes will have more growth where there is a higher concentration of oxygen and will have less growth at the bottom where they are using fermentation.

Bacillus subtilis is a mesophile, but it is used to determine the effectiveness of an autoclave. Explain why this is so.

Bacillus subtilis is used to determine the effectiveness of an autoclave because autoclaves are used to sterilize, meaning they remove microbes in any form, including the protective structures known as endospores. Endospores are highly resistant to all forms of disinfectant and are difficult to destroy. Bacillus subtilis is a type of species of bacteria that has the ability to form endospores, therefore, if an autoclave is able to effectively destroy these endospores then we know it is an effective physical method of control.

When comparing the Toilet Plate to the Mouth Plate, explain why one of the plates has significantly more growth than the other.

If both the Toilet Plate and the Mouth plate were incubated at 37 degrees Celsius, the bacteria from the mouth is used to body temperature since it came from the body, therefore it thrives and grows faster at that temperature. However, the bacteria found in the toilet water has a different temperature that its used to, and therefore grows slower at 37 degrees Celsius. Also, there is way more bacteria found in the mouth than in a toilet because toilets are frequently cleaned with antimicrobial agents, such as disinfectant that kill bacteria, so that may have caused less bacteria to be picked up.

How could you determine if one of the chemicals used in the experiment was bacteriostatic or bactericidal?

In order to determine if one of the chemicals used in the experiment was bacteriostatic or bactericidal, then you'd have to take the bacteria out of the media (plate) and put it into a new media. Once transferred and given time, if the chemical was a bacteriostatic agent, then you'd observe some regrowth with the bacteria because the chemical was only inhibiting the growth. However, if the chemical was a bactericidal agent, then there'd be no regrowth with the bacteria because it would've killed with the chemical agent.

Why is it important to perform a gram stain on an unknown sample of bacteria? How will this help you with the identification of the unknown?

It's important to perform a gram stain on an unknown sample of bacteria because you'll be able to identify the shape, arrangement and cell wall composition of the unknown bacteria. This will help you narrow down your choices of species of bacteria, because shape and arrangement is specific to the type of bacteria, as well as, having a gram positive or gram negative cell wall.

This is an example of

Staphylococci (cluster of grapes)

What were the results for the gram stains on: Unknown A (Streptococcus) Unknown B (Pseudomonas)

Unknown A (Streptococcus): Streptococcus had a gram positive cell wall (purple/blue), a cocci shape and a streptococci arrangement. Unknown B (Pseudomonas): Pseudomonas had a gram negative cell wall (red/pink), a bacilli shape and the arrangement was single bacilli.

Your lab partner found color pictures of a Gram stain of E. coli and S. epidermidis in the microbiology textbook. She has decided that she will not put E. coli and S. epidermidis on the same slide as her unknowns. Instead, she will make a smear of her unknown only and then look at the pictures in the book. Is this a good idea or not? Explain

No, this is not a good idea because the Gram stain of E. coli and S. epidermidis are used as controls to make sure that the Gram stain procedure was done correctly. If she doesn't put E. coli and S. epidermidis on the slide with her unknown then she won't know for sure if she performed the experiment correctly, and may get false results, which can then lead her down the wrong path when it comes to identifying what her unknown bacteria is. For instance, if she had the E. coli and S. epidermidis on the slide with her unknown and the E. coli was pink in color and the S. epidermidis was purple in color, then she'd know for sure that she stained the bacteria correctly and that the results of her unknown are also correct.

Your lab partner was looking at the smear he made with the 100x objective, but is running out of time to finish his observations. He asks you if he can clean off the slide and keep the slide in the slide box until next class period. Is this a good idea or not? Explain

No, this is not a good idea because he is using a smear that he made and not an already prepared slide with a cover slip, which prevents the bacterial cells from being wiped off. Since he is using the 100x and there is oil on the slide, cleaning the oil off of the slide would in turn, remove the bacterial cells as well. He would have to redo his smear, which he probably won't have time for next class period. And not only does he have to clean off the slide, but since he was using the oil immersion lens, he must also clean off the oil on the lens, using lens paper, or else the lens will produce a blurry image.

Using a simple stain, would you be able to identify the bacteria that cause Scarlet fever from the bacteria that cause dental caries? Explain why or why not and if you can't differentiate them with a simple stain what other method could you use?

No, you would not be able to identify the bacteria that causes Scarlet fever from the bacteria that causes dental caries using a simple stain, because the bacteria that causes Scarlet fever is Streptococcus pyogenes and the bacteria that causes dental caries is Streptococcus mutans, which means that both of these bacteria would be cocci in shape and have a chain-like arrangement, leaving no room for differentiation. In order to differentiate these two types of bacteria from one another, you can use Koch's postulates in order to see how these different types of bacteria cause different types of disease within a host.

This is an example of

Single bacilli (rod-shaped)

This is an example of

Streptococci (chain)

Another scientist wants to catalogue the microbes that live below the ice in antarctica. What type of culture conditions should be used to grow the microbes from that area?

When culturing microbes that live below the ice in antarctica, they need to be cultured at a temperature of about 15 degrees Celsius since they are used to colder temperatures and stored in a dark place.

An ecologist wants to catalogue the microbes that live in a hot spring. What type of culture conditions should be used to grow the microbes from that area?

When culturing microbes that live in a hot spring, they need to be cultured at about 50-60 degrees Celsius to ensure that the microbes are able to grow and thrive because the microbes are used to living in a hot environment.

When plates are crowded the colonies tend to be smaller, explain why

When plates are crowded, the colonies tend to be smaller because the bacteria are competing with one another in order to metabolize the nutrients within the agar and use it as fuel to grow and divide. If they are too close together then that means there isn't enough nutrients or fuel to go around, therefore, creating smaller colonies. This explains why the more spread out the bacteria is, the bigger the colonies are because they don't have to compete with other colonies for nutrients, and can continuously grow and divide, creating a bigger colony.

Using a simple stain, would you be able to identify the bacteria that cause MRSA from the bacteria that cause anthrax? Explain why or why not and if you can't differentiate them with a simple stain what other method could you use?

Yes, you would be able to identify the bacteria that causes MRSA from the bacteria that causes anthrax using a simple stain, because the simple stain would highlight the cellular shape of the bacteria. For instance, the bacteria that causes MRSA is Staphylococcus aureus, which means that its shape is cocci (circular), while the bacteria that causes anthrax is Bacillus anthracis, which means that its shape is bacilli (rod-shaped). Using a simple stain would also show the arrangement of the bacteria, and the bacteria that causes MRSA would be in a cluster arrangement, while the bacteria that causes anthrax would be in a chain-like arrangement because it is a streptobacillus bacteria. These differences in cellular shape and arrangement would clearly allow an observer to identify these two different types of bacteria from one another.

List two different observations that indicate there is a contaminating colony on a streak plate. a. b.

a. Colonies from the same bacteria type should have the same morphology, so if there is a different morphology from the morphology of the bacteria being cultured, then that indicates that there is a contaminating colony on a streak plate. For example, E. coli should have a circular shape, with entire, smooth edges and a mucoid texture. But, if you happen to see a filamentous shape and a hairy texture accompanying the E. coli, then that indicates fungal contamination. b. If I were to breathe on my plate too closely then there would be bacteria from my mouth on the plate, which would be in areas on the plate that the loop of bacteria did not touch. So you would be able to visibly see that the bacteria from my mouth is randomly dispersed on the plate instead of within the lines of the streak made.

Give two reasons why it is important to perform a streak plate on a sample of unknown bacteria

a. It is important to perform a streak plate on a sample of unknown bacteria because dilution and isolation of bacteria is needed to observe the colony characteristics, which are specific to the genetic makeup of an organism b. It is important to perform a streak plate on a sample of unknown bacteria because varying characteristics in colony morphology can be used to identify different types of bacteria

Give three different explanations why a student might have too much growth (and no isolated colonies) on her streak plate. a. b. c.

a. The student may have picked up too much bacteria on the inoculating loop, rather than just touching the bacteria slightly, making it more difficult to dilute out the bacteria onto the TSA plate. b. The student may have picked up more bacteria from the culture tube in between streaks, thus not diluting it at all, but adding more to the plate. c. The student may have forgotten to flame the loop in between streaks, so the leftover bacteria on the loop was not diluted.

You perform a gram stain on your unknown and observe pink cocci and purple cocci in your sample. Give two explanations why this could have happened.

a. This could mean that the culture was older and that some of the cells within that culture were already beginning to die, causing their cell walls to deteriorate. In gram positive cells, those dead cells would no longer have a thick peptidoglycan layer and instead would have a thin one, which means that the dead cells wouldn't be able retain the purple crystal violet-iodine complex during the selective treatment and would be dyed pink with the safranin. However, since the alive cells still had their thick layer of peptidoglycan, then they were able to retain the purple crystal violet-iodine complex during the selective treatment and remain purple. Thus, resulting in a mixture of pink and purple cocci. b. The result of purple and pink cocci could mean that during the selective treatment step, the decolorizer was added too long to the slide, causing holes in the cell wall and washing out some of the purple crystal violet-iodine complex in the gram positive cells, resulting in pink and purple cocci after the safranin step.

Colony

definition: a visible population of microorganisms originating from a single parent cell and growing on a solid medium.

Ubiquity

definition: something that is found everywhere and is present at all times

What arrangement is this?

diplobacillus

What arrangement is this?

diplococcus

Describe the procedure used in lab 2

procedure: - step 1: clean bench with disinfectant, label plate (initials, class, bacteria being cultured) and draw a "T" shape on the bottom of the plate (agar side) - step 2: sterilize the loop in bunsen burner until it glows orangey red and let it cool for 30 seconds - step 3: obtain the cultured tube and flame the mouth of tube - take sterile loop and just touch it to where there's bacterial growth - step 4: use the lid as a shield and streak the bacteria back and forth, using a zigzag motion above the "T" - step 5: flame the loop again to kill off all remaining bacteria on the loop - step 6: using the lid as a shield, take the sterilized loop and go back into the first streak once or twice, picking up bacteria, and streak the bacteria back and forth into the second area, using a zigzag motion - step 7: sterilize the loop in the bunsen burner until it grows orangey red and let it cool for 30 seconds - step 8: using the lid as a shield, take the sterilized loop and go back into the second streak once or twice, picking up remaining bacteria and streak the bacteria back and forth into the empty space, creating a 3rd streak - make sure to not go into the other two streaks - step 9: flame the loop again - step 10: perform this same procedure two more times with other types of bacteria - step 11: stack the three plates upside down and place in the incubator (37 degrees Celsius)

Describe the procedure used in lab 5

procedure: - step 1: obtain a clean slide(s) and label (initials and types of bacteria) the backside of slide with a wax pencil - step 2: aseptically smear prep the slide (the ends should act as controls -gram negative and gram positive for the middle) - step 3: air dry the smear and heat fix the slides with the bunsen burner flame - step 4: use a clothespin to lay the slide flat (bacteria facing up) on the slide holder, over the sink - step 5: primary stain: flood gram's crystal violet over the entire smear and let it sit for 1 minute - step 6: use a clothespin to angle to slide slightly downwards, start at the top edge of the slide and gently rinse off with deionized water - step 7: lay the slide flat on the slide holder over the sink - step 8: mordant: flood iodine over the entire smear and let it sit for 1 minute - step 9: use a clothespin to angle to slide slightly downwards, start at the top edge of the slide and gently rinse off with deionized water - step 10: selective treatment: use a clothespin to angle to slide slightly downwards and rinse with alcohol at the top edge of the slide until the run-off at the bottom of the slide is clear (decolorizes) and rinse with water - step 11: counterstain: flood the smear with safranin and let it sit for 1 minute - step 12: use a clothespin to angle to slide slightly downwards, start at the top edge of the slide and gently rinse off with deionized water - step 13: use bibulous blotting paper to gently pat the slide dry - step 14: examine the slide underneath the microscope by focusing up to the oil immersion objective - step 15: clean the microscope with lens paper and the slide with detergent

Describe the procedure used in lab 1

procedure: - step 1: label the bottom (agar side) of your plate with a sharpie (initials, date, sample used, and class) - step 2: take a sterile swab and swab the surface (if dry, wet the swab with deionized water) - moving and rotating the swab back and forth with a decent amount of pressure - step 3: transfer microbes to the plate (relatively gentle) - use lid as a shield to prevent the agar from getting contaminated by the microbes in the air - step 4: put plate in the incubator (37 degrees Celsius) upside down - can see label and microbes on agar are performing cellular respiration and releasing carbon dioxide and water vapor, causing condensation (don't want the condensation to drip down onto the colonies)

Describe the procedure used in lab 3

procedure: - step 1: make sure the microscope is plugged in, the power switch is on, and place microscope so that it is away from the edge of the counter - step 2: clean the microscope using optical lens paper (tissue paper) and lens cleaner - clean the objective lenses (high and dry and oil lens) and the stage - step 3: adjust the oculars to fit your eyes - want to see one circle of light - step 4: put the slide on the center of the microscope - step 5: turn light source all the way down and put it about halfway, then start with the lowest object lens (scanning - 4x) when focusing - step 6: focus the lens only using the coarse adjustment knob - move stage all the way up and slowly move down until object comes into focus - step 7: once in focus, switch to the 10x objective lens - step 8: only use the fine adjustment knob (fine tunes) to focus the object - step 9: once in focus, switch to the high and dry (100x) objective lens and use the fine adjustment knob to focus - step 10: clean the oil lens (1000x), have an open space over slide, and use oil immersion (only used on bacterial cells) - put one drop of immersion oil over light on the stage and gently slide oil lens over - step 11: clean off objective lens, stage and slide

Describe the procedure used in lab 9 (disk diffusion)

procedure: disk diffusion assay - step 1: obtain a Mueller-Hinton plate and one blood agar plate for each organism being tested (three) - step 2: label each of the plates with the appropriate bacteria and divide into four quadrants and label with different chemicals (use same orientation) - step 3: sterilize the loop - step 4: flame the mouth of the cultured tube and plate sample of bacteria - step 5: create a lawn of bacteria on the M-H and the blood agar - step 6: after creating a lawn, pour the different chemicals (spic and span, hydrogen peroxide, iodine and alcohol) into watch glasses - step 7: sterilize forceps (tweezers) and pick up a filter paper disk and saturate it in the chemical - step 8: place soaked filter paper disk (make sure it's not dripping) in the center of the quadrant that corresponds with the chemical and push it down slightly - step 9: wipe off forceps and repeat with different chemical for the same bacteria - step 10: when switching to different bacterial plates, make sure to sterilize the forceps (tweezers) - step 11: pick up a filter paper disk and saturate it in the chemical - step 12: place soaked filter paper disk in the center of the quadrant that corresponds with the chemical - step 13: repeat for all chemical and bacteria types - step 14: incubate plates at 37 degrees Celsius for 24-48 hours - step 15: after incubation, measure the diameter of each zone of inhibition and record the measurement in mm (the larger the zone = the more effective the chemical is)

Lab 3 Microscope: Describe the purpose of this lab

purpose: 1) learn the parts of the microscope 2) learn how to correctly clean the microscope 3) learn how to correctly focus the microscope 4) learn how to use oil immersion

Lab 4 Simple Stains: Describe the purpose of this lab

purpose: 1) observe individual cells 2) determine cell morphology - individual cells and their shape 3) correctly prepare a smear 4) correctly perform a simple stain

Describe the procedure used in lab 9 (kirby-bauer)

procedure: kirby-bauer assay - step 1: obtain one Mueller-Hinton plate for each organism being tested (three) - step 2: label each of the plates with the appropriate bacteria - step 3: sterilize the loop - step 4: flame the mouth of the cultured tube and plate sample of bacteria - step 5: create a lawn of bacteria on the M-H plates - step 6: place the antibiotic disk dispenser over the plate and slowly slide the lever to release the disks (8 individual disks) - step 7: use forceps to gently press down on each disk to ensure they don't fall when the plate is placed upside down - step 8: incubate all the plates at 37 degrees Celsius for 24-48 hours - step 9: after incubation, measure the diameter of each zone of inhibition and record the measurement in mm - step 10: use a table to determine if the bacteria is resistant, intermediate or susceptible to each of the antibiotics

Describe the procedure used in lab 4 (17 steps)

procedure: smear preparation - step 1: make sure the glass slides are clean (free of any oil) - wash with detergent and dry with a paper towel - step 2: use a clothespin to hold the slide, use a sharpie and draw two lines to seperate the three different bacteria (unknown and controls) and label the the slide with initials and types of bacteria being used - step 3: broth culture: mix the culture by swirling the tube gently, flame the loop, touch the loop to where there's growth and apply one or two loopfuls of the culture to the center of the slide - step 4: use a circular motion to spread the bacteria evenly, until it is about the size of a dime - step 5: lawn or colony: flame the loop and use the water dropper to drop 1-2 drops of deionized water to the loop and put the water droplets on the center of the slide - step 6: flame the loop and transfer a small amount of bacteria from the solid medium to the water - step 7: use a circular motion to spread the bacteria evenly on top of the water droplet, until it is about the size of a dime - step 8: let the slide air dry for about 10 minutes- set it on a paper towel and move it out of the way (do not blow on it or wave it in the air) - step 9: once air dried, the smear should appear as a thin, translucent layer, almost like a water spot - step 10: heat fix the slide (this ensures the the bacteria stick) - use a clothespin to hold the slide and rapidly pass the slide (bacteria side faced up) 1-2 times over the outer flame (bottom of slide should fog up from the heat) - step 11: lay the slide with the bacteria side facing up on a slide holder above the sink and use a dropper to flood the smear with Gram's Crystal Violet and stain the entire surface of the smear and let it sit for 1 minute - step 12: after a minute, hold the edge of the slide with a clothespin and angle the slide downwards above the sink - step 13: used deionized water and and gently squirt the water at the top of the angle, allowing the water to gently flow down the slide and wash off the stain, without washing off the bacteria - step 14: after the excess stain has been washed off, put the slide inside bibulous blotting paper and gently press down on the outside of the paper a few times to blot and dry off the slide - do not wipe across the slide or it will remove the bacteria - step 15: after the slide has been gently dried, examine the slide underneath the microscope and focus each objective until you get to the oil immersion objective lens and record the results - step 16: after recording the results, clean off the slide using detergent and a scrub brush to remove the bacteria and the stain - step 17: use ethanol to remove the sharpie writing and dry the slide and put it back in the used slide box

Describe the procedure used in lab 8 temperature requirements

procedure: temperature - step 1: obtain six TSA plates - step 2: label two "4 degrees Celsius, two "25 degrees Celsius, two "37 degrees Celsius and two thick plates "55 degrees Celsius" - step 3: one of each type of plate will be divided into 3 equal sections and labeled with three different types of bacteria, while the other 4 plates will be labeled and streaked with S. marcescens - step 4: use a sterile inoculating loop and touch it to bacterial growth in tube - step 5: make a straight streak in one of the three quadrants that corresponds to the bacteria being inoculated - step 6: repeat with all bacteria (except S. marcescens must be streaked) - step 7: place all of the TSA plates in the appropriate temperature turned upside down

Describe the procedure used in lab 8 oxygen requirements

procedure: thioglycollate media - step 1: obtain thioglycollate media for each organism being tested - step 2: label all the tubes with the organism being tested - step 3: use a sterile (use bunsen burner) inoculating needle - step 4: flame the mouth of the culture tube and touch the needle to bacterial growth (solid media) - step 5: stab the inoculating needle all the way down the center of the thioglycollate media (do not mix it b/c it'll introduce more oxygen) - step 6: place in the incubator (37 degrees Celsius) procedure: gas pak jars - step 1: obtain one plastic TSA plate (plastic fits in the jar)and one glass TSA plate for each organism - step 2: label the plastic TSA plate "gaspak" (no oxygen present) and label the glass TSA plate "incubator" - also make four quadrants and label the different types of bacteria (label in same order) - step 3: sterilize an inoculating loop and let it cool for 30 seconds - step 4: flame the mouth of the culture tube and touch the inoculating loop to the bacterial growth - step 5: make a straight streak in one of the quadrants, that corresponds with the bacteria being inoculated (do for incubator and gaspak) - step 6: sterile the loop and repeat with different bacteria - step 7: incubate for 24-48 hours and compare growth to determine oxygen requirements

What arrangement is this?

staphylococcus

What arrangement is this?

streptobacillus

What arrangement is this?

streptococcus

What arrangement is this?

tetrad