Practicum 1 - Microbio

What does septic mean?

rotting; putrification

What are the agents that cause septic?

Microorganisms

What does aseptic mean?

Without rotting/bacteria/microorganisms

What are some examples of aseptic techniques in lab to prevent contamination?

1. Media

2. Bunsen Burner

What is media?

a liquid or solid nutrient suspension

How is media sterilized?

Using an autoclave that uses high temperatures (moist heat/steam) and pressure

When do we use media?

Not opened until ready for use (quick open or clam shell and no lids on the table)

What is a Bunsen burner?

heat bomb

What is a bunsen burner used for?

flaming tubes after opening and before closing

How do you know tools are sterile when using a Bunsen burner?

tools turn orange

What are the 4 forms of media?

1. Broth

2. Plate

3. Slant

4. Deep

What is a broth and where is the bacteria found?

liquid media in a tube using an inoculation loop to place bacteria in media

What are the advantages of a broth?

• easy to transport via tube rack

• low contamination b/c of small opening and able to pass through fire

• fast group up to around 48 hours because nutrients go in and waste comes out, but it reaches a capacity when surrounded by waste

What are the disadvantages of a broth?

• no isolation, unable to identify if there are 2 or more bacteria because it appears cloudy/turbid, no differences

• no sustained growth (capacity of 48 hours)

What is a plate and where is the bacteria located?

a petri dish with a solid gel layer on top and with a lid; bacteria is located on top of the gel layer with an inoculation loop

What are the advantages of a plate?

• only form of media that can achieve isolation and differentiate between 2 or more bacteria

• slow, sustained growth since nutrients come from the bottom

What are the disadvantages of a plate?

• high contamination because there is increased surface area

• can dry out because there is increased surface area and a thin gel layer

• harder to transport since lids come off easily and they must be stacked

What is a slant and where is the bacteria placed?

solidified media at an angle in a tube where the bacteria is on a slanted surface using an inoculation loop

What is a slant a combination of?

A broth and plate

What are the advantages of a slant?

• easy to transport (tube rack)

• low contamination (small opening and able to pass through fire)

• slow, sustained growth (solid surface with nutrients coming from the bottom)

What are the disadvantages of a slant?

no isolation

What is a deep and where is the bacteria placed?

solid media in a tube with a flat surface; bacteria goes in media using an inoculation needle

What are the uses of a deep?

• cultures organisms that prefer low oxygen

• Detects motility (bacteria that move have flagella)

What is a flagella?

propeller that spins in different directions and can allow bacteria to move

What are the two types of motility?

1. Nonmotile

2. motile

What are the results of the two types of motility in a deep media?

• Nonmotile bacteria result in bacterial growth where the stab is tracing one line

• Motile bacteria swim through the media and create a vortex

Define an aseptic transfer goal

transfer bacteria from one tube to another without getting it contaminated

Steps of an aseptic transfer

1. Hold inoculation loop in your dominant hand

2. Flame loop until orange to sterilize

3. Let loop cool for 20 seconds

4. Grab first tube with non-dominant hand

5. Remove cap using dominant hand pinky (move tube away from cap)

6. Flame tube

7. Dip loop into tube to obtain culture

8. Remove loop

9. Flame tube

10. Recap first tube (move tube towards cap)

11. Place tube down on rack

12. Grab second tube with non-dominant hand

13. Remove cap using dominant hand pinky

14. Flame tube

15. Dip loaded loop into tube to deliver inoculum

16. Remove loop

17. Flame tube

18. Recap second tube

19. Place tube down on rack

20. Flame loop until orange to sterilize

Tools found in the lab and their functions

• Inoculation loop – transfer bacteria, inoculate media

• Inoculation needle – inoculate deeps

• Forceps – grasp small items

• Test tube clamp – grasp hot test tubes

• Clothes pins – grasp microscope slides

• Microscope slides – prepare smears and stains

• Test tube rack (blue rack) – hold and transport test tubes

• Bibulous paper – absorbs liquid from wet microscope slides

• Lens paper – clean microscope lenses

• Ruler – measure short distances

• Bunsen burner – sustain gas flame, sterilization

• Striker – initiate spark

• Water bottle – rinsing slides during stains

• Cavicide – surface disinfectant

Safety precautions taken in lab

• Hair tied back

• wear lab coat

• closed-toed shoes

• know where shower/eye shower is

• where evacuation routes are

• carrying around tubes with a blue rack

• knowing which bins are for what (gray bin in the hood is for trays, tubes go in fume hood bins, slides and pipettes go in red hazard foot bin)

• handwashing before and after lab

• Cavicide on table

• use the correct wipe materials (Kim wipes for spills, lens paper for microscope lens)

• no food or drink

• follow instructions

• report accidents, cuts or injuries

Lab personal protective equipment

• Goggles

• Lab coat

• Gloves

• Close toed shoes

What does it mean when microbes are ubiquitous?

found everywhere

What are the 4 categories of microbes?

1. resident flora

2. transient flora

3. opportunistic pathogen (opportunist)

4. Pathogens

What are resident flora?

microbes that live in or on you, colonizing your body and living as residents via a symbiotic relationship

What is a symbiotic relationship?

close and long term interaction between two species; living together

What are the types of symbiotic relationships?

1. commensalism

2. mutualism

What is commensalism?

one organism benefits and the other is indifferent

What is an example of commensalism?

an animal sitting under the shade of a tree where the animal benefits and the tree is indifferent

What is mutualism?

both organisms benefit

What is an example of mutualism?

bacteria (E. coli) in our gut getting free food, water, nutrients, temp, helping us with digestion by breaking down food we are unable to digest, and vitamin absorptionAn example of mutualism is when bacteria such as $E. coli$ in our gut receive nutrients and help us with digestion and vitamin absorption.

What is transient flora?

microbes that come in contact with us, but leave because our bodies are not the right environment; on the body for a short time and do not colonize, but move

What are considered normal flora

resident flora and transient flora

What is sometimes considered normal flora

opportunistic pathogen (opportunist)

What is an opportunistic pathogen (opportunist)?

only causes disease when the opportunity presents itself—conditions where they can take advantage; do not cause disease in healthy individuals

What are the conditions opportunists take advantage of?

• weak immune system

• cuts/breaks in skin

What is a fungal example of an opportunist?

Candida albicans

What is Candida albicans?

a type of yeast/mold that causes yeast infections and is found on males and females in the mouth and urogenital regions

What is a bacterial example of an opportunist?

Staphylococcus aureus

What is Staphylococcus aureus?

bacteria that causes staph (skin) infections, digestive issues, and produces heat toxins

What is secondary yeast infection?

taking antibiotics that wipes out bacteria in and on the body, killing off “normal” bacteria, which allows Candida albicans to spread because it is a fungus

What is the percentage of people that have Staphylococcus aureus on their body and it is considered normal if healthy?

24%

What conditions do Staph infections occur in?

cut, popped pimples, break in skin

What are pathogens?

disease causing microbes

What is required when culturing bacteria?

media

What are the two categories of media?

1. chemically defined media

2. complex media

What is chemically defined media?

exact chemical composition is known down to molecular and atomical percentages

What is complex media?

exact chemical composition varies slightly with each batch

What are examples of complex media

nutrient broth, agar, Tryptic Soy Agar (TSA)

What different types of nutrient extracts can complex media at least contain?

1. yeast extract

2. beef extract

3. peptone (partially digested proteins)

What is agar

marine algae/seaweed extract that acts as a solidfying agent

What are the properties of agar?

1. most microbes cannot degrade it and get nutrients from it, so it remains solid

2. liquifies at 100C and it stays liquid until 40C

How and why do we label plates?

• name—date—media—group—sample site

  • labeled on the bottom of the petri dish because lids can be swapped out accidentally

How and why do we incubate plates?

• incubated at 38C upside down for 48 hours or at 25 degrees upside down for a week

• incubated upside down to avoid evaporation and condensation of the fluid and to see the label

Environments with the greatest amount of microbes with examples?

Warm and moist environments

• bottom of shoes, floor, skin, water bottles

Environments with the least amount of microbes with examples?

Cold and smooth environments

• clock, whiteboard

What are the two used products for handwashing?

1. soaps and detergents

2. hand sanitizers

What is the function of soaps and detergents?

act as surfactants

What are surfactants?

wetting agents that decrease surface tension that breaks hydrogen bonds and spread water to wet more areas, but emulsifies fats and oils on the skin when combined with scrubbing allowing them to be washed away

What is the result of using soaps and detergents for handwashing?

mechanical removal of bacteria on skin with proper rinsing

What is the main intention of soaps and detergents?

NOT to kill bacteria, but some do die

What is the main active ingredient of hand sanitizers?

different alcohols, but most of the time ethanol or isopropanol

What is alcohol effective against?

bacteria, fungi, viruses

What is alcohol ineffective against (makes hand sanitizers only 99.99% effective)

(endo)spores

What is the function of hand sanitizers?

denature proteins and disrupt cytoplasmic membranes

What are proteins?

long chains of amino acids (polypeptide) folded into a ball

What happens when proteins denature?

unfolded to original shape, losing its function

What is the result of using hand sanitizer?

kills microbes

What is the optimal range for percentage of ethanol in hand sanitizers?

62 to 70%

Why are hand sanitizers not 100%?

1. denaturing proteins requires water

2. the more ethanol, the fast it evaporates and less contact time, reducing its effectiveness

Commonly missed areas of handwashing

• wrists

• fingers

• in between fingers

• back of hands

• fingernails

How much bacteria/mL of saliva does the mouth have?

millions

What is the predominant genus in everybody’s mouth and is normal flora?

Streptococcus

What are the two major organisms of Streptococcus?

1. Streptococcus mutans

2. Streptococcus sanguinis

If there is a lot of Streptococcus mutans, what is there increased susceptibility to?

cavities

What does Streptococcus mutans produce from sucrose and why?

produces sticky polysaccharides because Streptococcus hydrolyzes sucrose

What is Streptococcus sugar of preference? Describe it

sucrose — a disaaccharide

What does sucrose break down to form and how?

Sucrose is hydrolyzed by adding water, forming glucose and fructose (monosaccharides)

What does glucose form?

forms long chains called dextran

What is dextran, what does it do, and what does it lead to?

• sticky carbohydrate chain that surrounds the bacteria

• enables bacteria to stick to teeth

• leads to plaque

What is plaque?

masses of bacteria, dextran, and debris adhering to the teeth

What does fructose form? How?

lactic acid through fermentation

What does lactic acid cause and result in?

• erodes/degrades the enamel to the dentin, feeding on it

• results in cavities/dental caries because of the high concentration and close proximity

• Streptococcus then feeds/grows on dentin and tooth pulp

What is the difference between sugars and sugarless candies?

Sugarless candies contain sugar alcohols to provide a fake sweet flavor, which do not form dextran that can cause plaque. Sugar candies can cause plaque because of the sucrose.

What are two examples of sugar alcohols?

sorbitol and mannitol

How can sugarless candies still cause dental caries, but not as much as sugar candies?

the sugar alcohols can still ferment to produce lactic acid, increasing tooth erosion if plaque is already present

What is the media that tells predisposition to dental caries?

Snyder Agar

What does Snyder Agar contain?

sucrose, bromcresol green, yeast extract, NaCl, agar

What is Bromcresol Green and what does it indicate via its colors?

• pH indicator

  • yellow = acidic (cavity)

  • green = neutral

  • blue = basic

How is Bromcresol Green used to identify dental caries?

Fructose, broken down from sucrose, ferments to produce lactic acid and results in a cavity; saliva sample is placed into the molten agar and is incubated for 5 to 7 days; if media is yellow, then there is a cavity and the first statement truly happened

What kind of microscope is used in this class?

Compound brightfield microscope