Microbiology Lab Practical Midterm

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What is the purpose of a four quadrant streak plate?

To isolate bacteria or bacterial colonies.

What are the different categories of colony characteristics?

Form
Elevation
Margin

Form

referes to the basic shape of the colony, for examples circular, irregular, or filamentous.

Elevation

referes to the cross-sectional shape of the colony

Margin

refers to the magnified shape of the edge of the colony.

What are the three types of bacterial growth characteristics in broth?

Pellicle
Sediment
Turbidity

Pellicle

Is a thin film of bacteria growing at the surface of nutrient broth, which means it is a strict aerobe (requiring oxygen for growth).

Sediment

Bacteria growing at the bottom of a broth, could be a facultative anaerobe or an anaerobic bacteria (a bacteria that grows without oxygen).

Turbidity

is growth of a bacteria that occurs throughout the broth, such as a facultative anaerobe (a bacteria whose growth is accelerated by the presence of oxygen).

Simple Stain

Has one basic dye with a positive charge.
It shows the morphology of the bacteria meaning the shape and arrangement of the bacteria.

Gram Stain Includes?

Primary Stain
Mordant
Decolorizer
Counter Stain

What is is the primary stain used in gram stain?

Crystal Violet

What is the mordant used in gram stain?

Iodine

What is the decolorizer used in gram stain?

Alcohol or acetone

What is the counter stain used in gram stain?

Safranin

Gram positive bacteria will be what color after being treated with a gram stain?

Purple

Gram negative bacteria will be what color after being treated with a gram stain?

Pink

What are the two types of spore forming organisms?

Clostridium and Bacillus (Endospores)

What type of stain do Clostridium and Bacillus require?

Spore Stain which uses Malachite Green and Safranin. Water is decolorizing agent

What type of organisms require Acid Fast stain?

Mycobacterium (a gram positiver bacillus containing mycolic acid).

What dyes are required for an acid fast stain?

Carbolfuchsin
Acid Alcohol
Methylene Blue

What color do acid fast bacteria turn after being treated with acid fast stain?

A deep red

What color do non-acid fast bacteria turn after being treated with acid fast stain?

Blue

What happens to the capsule of an organism after a negative or capsule stain?

The capsule remains clear because no dyes can stain a capsule.

When put in a motility medium how does one determine if the organism is motile or non-motile?

Non-motile organisms will only grow along the puncture line of the medium.

Motile organisms will grow and spread out form the puncture line of the medium.

What is a fomite?

A fomite is an inanimate object or substance capable of carrying infectious organisms, such as germs or parasites, and spreading them to living things.

Disinfection

Is the partial irradication of microorganisms.

What types of tubes are used to determine if an organism is a ferementor of carbohydrates?

Durham Tubes (designed to catch gas bubbles).

What is the pH indicator used in determining the fermentation of carbohydrates?

Phenol Red

What does it mean when a durham tube is red?

It means that it is basic and a negative result for carbohydrate fermentation.

What does it mean when a durham tube is yellow?

It means it is acidic and a positive result for carbohydrate fermentation.

What does it mean when there appears to be air in the Durham tube?

It means that the carbohydrate fermentation produced gas bubbles which were trapped there.

What medium is used to determine the digestion of starch?

The test enzyme amylase
Iodine

What does a positive reaction concerning starch digestion look like?

Positive for Amylase is a halo visible around the area where starch is utilized.

What enzyme is used in DNA digestion?

Enzyme Dnase - which breaks down long chains of DNA, and uses Methyl Green dye.

What does a positive Dnase result look like?

A clearing around the streak on the streak plate. A clear halo.

What does catalase mean?

To break down into hydrogen-peroxide into H2O and O2.

What represents if catalase is present?

A positive result is determined by bubbles.

What if catalase is absent?

No bubbles will be present, meaning a negative result.

Is streptococci catalase positive or catalase negative?

Catalase negative

Is staphlococci catalase positive or negative?

Catalase positive

When is the black precipitate formed?

H2S gas production during the reaction

What does SIM stand for?

Sulfide Indole Motility

What enzyme is used in indole production?

Tryptophanase - which breaks down tryptophan into indole, pyruvic acid and ammonia.

What reagent is used to test for indole production?

Kovac's Agent

If indole is present and Kovac's reagent is added what will occur?

A positive reaction of a red ring forming at the top of the medium or broth.

If indole is not present and Kovac's reagent is added what will occur?

A negative reaction of an amber or greenish ring at the top of the medium or broth.

What enzyme is used to determine urea digestion?

Urease

What indicator is used to determine urea digestion?

Phenol Red

What color is phenol red at basic pH?

Red

What color is phenol red at acidic pH?

Yellow

What color is phenol red with the presence of urea?

Hot pink due to the ammonia released from urea.

What is antiseptic used on?

Living cells and tissues

What is disinfectant used on?

Inanimate Objects

What are the modes of transmission of infectious diseases?

1. Skin to Skin Contact (Cold Sores)
2. Body Fluids (Mono)
3. Air (Sneezing/Coughing/Influenza)
4. Fomites (Cellphones/Door Handles)
5. Oral/Fecal (E.Coli)
6. Arthropage Vector Borne (Tick: Lyme Disease)
7. Transplacental (Syphillus)

What is the most effective way to prevent diseases?

Washing hands with soap and water.

What is the importance of the aseptic technique?

A universal technique used in laboratories to prevent the contamination of the work area, helping transfer organisms without contaminating the surrounding areas.

What is a broth medium?

A liquid medium containing all the nutrients that a bacteria can utilize for growth.

Where do you label a Petri plate?

A small notch on the corner. with your initials, station number, and bacteria used.

What is the name of the solidifying agent used most successfully in bacterial nutrient media?

Agar

A colony consists of how many bacteria?

One Million

What is common between Clostridium and Bacillus?

They are both endospores

In acid fast staining, what is the name of the coating that protect the cell wall from stain penetration?

mycolic acid

List three terms that describe bacterial cell shapes?

Coccus
Bacillus
Spirilum

Gram Positive Bacteria

1. Thick PTG wall (30 layers)
2. Susceptible to Penicillin
3. Susceptible to Lysosomes
4. Purple stained

Gram Negative Bacteria

1. Thin PTG wall (2-3 layers).
2. Not susceptible to penicillin
3. not susceptible to lysosomes
4. Red stained

What is the purpose of immersion oil?

Prevents the loss of light rays due to diffraction and improves resolution.

What is resolving power?

the ability to distinguish two points as distinct, separate objects rather than one blurred image.

Who invented the microscope?

Anton Van Leeuwenhoek

Name the structure used for motility in both eukaryotes and prokaryotes?

Flagella

List three bacteria and diseases caused by endospore forming bacteria.

Clostridium tentai - Tentus
Clostridium Botulinum - Botulism
Bacillis Anthraces - Anthrax.

What is the difference between a negative stain and a capsule stain?

A negative stain is a one step process in which the background is stained instead of the organism.

A capsule stain stains the protective coating of the bacteria agains the negative background stain.

List the bacteria and diseases caused by bacteria that possess capsules.

S. Pneumonia - pneumonia, meningitis, otitis media
E. Coli - UTI, diaherria, gastroenteritis.
K. Pneumonia - pneumonia
N. meningitis - meningitis

Is acid fast stain a simple of differential stain?

Differential

Name the primary and counter stain in the acid fast stain.

Primary - Carbolfusin
Counter - Methyl Blue

List all the reasons for studying biochemical characteristics of bacteria.

It allows us to identify genus and species.

What is the end product of carbohydrate fermentation?

Acid or Acid and Gas Production

Name the enzyme that breaks down the polysaccharide into smaller carbohydrates?

Amylase

What are the three basic forms of media?

broth, semisolid, and solid

What is the function of broth media?

-liquid at room temp and incubation temp
-beef bouillon (beef extract, peptone, and water)
-nutrient broth (urea)

What is the function of the semisolid media?

-jelly like consistency
-too soft to form a slant, but more solid than a broth
-SIM medium contains beef extract, peptone, peptonized iron, sodium thiosulfate and 3g of agar
-3-5g/L of agar

What is the function of the solid media?

-solid at room temperature and normal incubation temps
-15g/L of agar
-agar deeps, slants (TSA slant), and plates are options

What is difference between solid and semisolid?

-solid has 15g/L of agar
-semisolid has 3-5g/L of solid

What is the difference between complex and chemically defined medium?

-complex: contains organic components; most medias used in lab
-chemically: contains inorganic chemicals like glucose, Simmon's citrate agar

What are the 4 types of sterilization?

filter, gas, dry-heat, and autoclaving

Filter sterilization

-clear solution is forced through a membrane that contains 0.2micron pores
-filters all bacteria but not viruses
-urea broth is sterilized this way

Gas sterilization

-ethylene oxide is used for things that would melt in the autoclave
-IV tubing, catheters, etc

Dry-heat sterilization

-heating of glass or metal to 160-170 C for 2+ hours
-not satisfactory for paper or liquid products

Autoclaving

-sterilizes 95% of items in this lab
-121 C at 15psi for 15+ minutes

Incubator

have thermostats that can be set for a specific temp. Usually between 30 and 37 C.

Celsius

(F - 32)/1.8

Fahrenheit

1.8C + 32

Common Temps (freezing point of water, room, incubator, body, boiling water at room temp and autoclave at 15psi)

water freezes: 0 C & 32 F
room temp: 22-25 C & 71.6-77 F
incubator: 30-37 C & 86-98.6 F
body temp: 37 C & 98.6 F
water boils: 100 C and 212 F
water boils at 15psi: 121 C and 250 F

Total Magnification

-eye piece 10X
-low 10X
-high 40X
-oil immersion 100X
total mag= eye piece x objective
1000X = 10X x 100X

Voltage Control

adjusts the intensity of the light transmitted to the specimen

Course adjustment knob

larger knob; moves stage up or down to bring specimen into focus

Fine adjustment knob

smaller knob; makes small changes in working distance

Condenser adjustment knob

condenser should be on "O" for bright; the adjustment knob controls the height of condenser btwn stage and light source

Iris diaphragm annulus

controls amount of light transmitted to the specimen

What does parfocal mean?

One a specimen has been focused on in 10x the microscope has the ability to switch to higher magnification with a minimum focal adjustment.

Working distance

distance from top of slide to the objective lens

Resolving power

lens' ability to distinguish between two separate points. smaller the resolving power is the better the lens

What are the basic bacteria shapes?

Coccus (circular)
Bacillis (rod-like)
Spirilum (spiral shaped)

What are the typical bacteria arrangements?

Staphylo - a cluster of bacteria
Strepto - a chain of bacteria
Tetrad - a square cluster of bacteria
Sarcina - a stack of square cluster bacteria.

Staphylococcus aureus

staphylo- clumps of cells like grapes
coccus- sphere shape

Bacillus subtilis

-rod shaped bacteria that forms chains
-gram +
-found in soil
-arborescent TSA slant

Escherichia coli

small bacillus/rod in diplo arrangement or random

Rhodospirillum rubrum

sprial shaped bacterial that don't aggregate in any fashion (random)

Clinical signs

objective criteria used to assess a disease state (temp, rash, BP, etc)

Clinical symptoms

subjective criteria a patient uses to describe their disease state (headache, pain, fatigue)

Communicable disease

a disease that spreads from one host to another by direct OR indirect means (ie: cold or flu)

Noncommunicable disease

a disease that is not spread from one host to another (tetanus)

Endemic

a disease that is always present in a population at a low level (west nile in texas)

Common source epidemic

the sudden increase in the number of individuals infected by a specific disease followed by a rapid decline in the number of cases (food poisoning...not contagious)

Propagated epidemic

slow, gradual increase in the number of individuals infected by a specific disease agent followed by a peak and then a slow decline in the number of cases. Disease is contagious...measles, chicken pox

Incidence

the number of people who become ill from a particular disease in a time period

Morbidity

(the number of people ill from a disease)/(the number of people exposed)

Mortality

(the number of people who die from a disease)/(the number of people who were ill)

Outbreak

sudden, unexpected occurrence of a disease

Pandemic

worldwide epidemic

Reservoir

organism or object that provides a pathogen with living conditions until it can transmit (bird carrying west nile)

Vector transmission

insect that transmits a disease from host to host (mosquito transmits west nile)

Vehicle transmission

fecal-oral transmission: food poisoning and intoxication
airborne transmission: spread of agents more than 1m from source (TB and measles and cold)

Direct contact transmission

person to person (std, colds, flus, measles)

Indirect contact

involves the use of a nonliving intermediate (FOMITE) like dishes and silverware

Droplet transmission

different than airborne; involves the droplet to be expelled less than 1m from source (flu and measles)

Normal flora

microbes that commonly inhabit a certain environment (e. coli in gut)

What elevations can a bacterial colony have?

Raised
Convex (dome)
Flat
Umbonate (looks like a fried egg)

What forms can a bacterial colony have?

Circular
Irregular
Filamentous
Rhizoid

What margins can a bacterial colony have?

Entire (circular)
Undulate (wavey)
Filiform (filaments)
Curled (curdled apperance)
Lobate (big lobes)

What consistency can a bacterial colony have?

Dry - ground glass appearance
Matte - flat paint look
Shiny - glossy
Mucoid - moist

What qualities of the skin protect from infection?

high salt content (sweat), low pH (acidity), fatty acid covering by sebaceous gland, and lysozyme secretion from sweat gland

Who first figured out we should wash our hands?

Dr. Sammelweis

Who is the father of antiseptic surgery and what did he do?

Joseph Lister and he used carbolic acid (phenol) to prep surgical sites

What is MRSA and where is it found?

Methicillin resistant Staphylococcus aureus & found in nasal passage

CA-MRSA vs HA-MRSA

healthcare associated MRSA and community associated MRSA

What antibiotic can you use on MRSA?

Oxacillin

What is coagulase?

exoenzyme secreted by Stapy

What is an autotroph?

uses inorganic C

What is a heterotroph?

uses organic C

Photo vs Chemoautotroph?

photo- uses sun as energy
chemo-oxidation of chemicals as energy

What are the temperature bacteria classifications?

psychophiles: frigid, -5-20 C
psychotrophs: grow 0-30 C; food spoilers
mesophiles: 20-45 C; human pathogens
thermophiles: 40-60 C; hottt

What is a pure culture?

one species only (Mixed is more than one)

What is subculturing and what else is it called?

transfer organism from one medium to another; inoculation

What does asepsis mean?

without organism; so it what we want in aseptic technique so there is no contamination

How do you grow pure colony?

streak plate to grow single colony; TSA slant to grow that pure colony; gram stain to test for purity

What is the inoculating needle or loop for?

needle: from a slant or broth to deep
loop: to make a streak plate
(either is good to transfer TO broth)

Micrococcus luteus

-gram +
-tetrad of cocci
-BRIGHT yellow colonies on TSA plate
-TSA slant = filiform
-broth = sediment
-normal flora

Pseudomonas aeruginosa

-random arrangement of small rods
-pathogen (opportunistic)
-TSA slant will turn GREEN from pyocyanin but colony will be pinkish
-AEROBIC and MOTILE
-corn tortilla smell

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