Micro Lab M1

What is proper aseptic technique?

1) label plate or tube 2) obtain sample you are transferring 3) if transferring to or from a tube, be sure to flame the lip after every step 4) open sample (flame the loop stem to tip) get some of the sample with loop 5) flame lip if tube and return sample 6) streak obtained sample on plate/slant or place in broth (flame loop of new tube) 7) continue streak or flame lip and loop and finish ALWAY FLAME LIPS AND LOOPS TO STERILIZE

what are 3 scenarios in which phase contrast microscopy would be preferable over bright field microscopy

1) provides better detail of structures 2) doesn't need a stain so organisms can be alive 3)can see organelles and endospores without distortion, can determine the size

what are two factors that affect resolution?

1)The wavelength of light (shorter wavelengths are able to resolve smaller objects) Electron microscopes resolve the best bc has very short wavelength) 2)The numerical apertures of the condenser and objective lens (measure of a lens ability to gather light) D=min distance at which two points can be resolved

Simple/chemically defined media

1. A chemically defined medium is one prepared from purified ingredients and therefore whose exact composition is known. 2. Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar are considered as basal medium. These media are generally used for the primary isolation of microorganisms. can also be considered selective since we control what is in there and what is not ex) know the exact concentrations of everythinging in the media, so blood agar

Complex media

1. Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. 2. Contents are not normally known Just putting a little of everything in there to see growth since we might not know exactly what it needs to grow yet

Eukaryotic cells

1. Have a nucleus that is surrounded by a complex nuclear membrane that contains multiple rod shaped chromosomes 2. All plant and animal cells are eukaryotic 3. Larger in size 4. Compartmentalizes various chemical processes witin the different areas of the cell using membrane bound organelles 5. Ribsome is 80S

Prokaryotic cells

1. Lack a nucleushave single circular chromosome located in a nucleoid 2. Have two domains (archea and bacteria) 3. Lack membrane bound organelles, but have inclusion that compartmentalize their cytoplasm 4. Group together in distincitive arrangements depending on the plane of cell division 5. Have a cell wall in combination with cytoskeletal elements (found in most) 6. Chromosomes are typically circular, haploid (unpaired) and not bound by nuclear membrane. DNA is associated with nucleoid region (has nucleoid associated proteins that organisze the chromosmes=similar to histones in euk) 7. Contain extrachromosomal DNA in circular plasmids that often carry antibiotic resistance 8. Ribosomes are 70S 9. Have inclusions for nutrient storage 10. Generally vegetative cells but can form endospores

what is the gene that Woese and Fox used to construct the current 3 domain model of the phylogenetic tree of life

16S r RNA of the ribosome

what is the proper labeling of microbial specimens

Plate=labeled along the edge of the base (not the lid since it could get lost) tube= label on piece of tape attached to the tube (not the cap) put Name, Date, Lab Section, Organism, Plate type, and incubation time/temp

predict the changes to a given microbial community if specific environmental factors are altered

• If environmental factors changed, it could alter the growth of a microbe if no longer in prime environment to grow -- think of Clostridum Difficle and how in a low microbial environemnt, it flourishes since it no longer has to compete for nutrients and thus the disease will occur (oppurtunistic pathogens)

why would an acidic dye react differently with a given specimen than a basic dye

• It will either be attracted to or repeled by the specimen depending on its overall charge • Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains

what are the 7 basic categories of colony morphology

• Size (large small) • Shape( round, irregular, punctiform) • Margin (entire, undulate, lobate, filamentous, rhizoid) • Surface (smooth , rough, wrinkled, shiny, dull) • Texture (moist, mucoid, butyrous, dry) • Elevations (flat, raised, convex, pulvinate, umbonate) • Color (opaque, translucent)

why is it best to adjusst the focus of the light microscope with the coarse focusing knob before using the fine focus knob

• So that you can get the stage close up to the lenses, then use the fine knob to make the image clearer, if you try to use the course then you could possibly get to close by accident and crack the slide that you are looking at once at an adequate height with the 10x lense then will be good for other lenses so only need the fine knob

Theory of Spontaneous generation and why peoople once accepted it as an explanation for existence of certain types of organisms

• The Greek philosopher Aristotle (384-322 BC) was one of the earliest recorded scholars to articulate the theory of spontaneous generation, the notion that life can arise from nonliving matter. Aristotle proposed that life arose from nonliving material if the material contained pneuma ("vital heat"). As evidence, he noted several instances of the appearance of animals from environments previously devoid of such animals, such as the seemingly sudden appearance of fish in a new puddle of water.

YTA media

Yeast-Tryptone Agar (complex media) --rich but lacks sugar --Non-selective for bacteria --general media, has what fastidious bacteria needs -- rich, nutritious medium for the recovery of a wide range of bacteria, yeast, and molds. enrichment

YTB media

Yeast-Tryptone Broth --also contains distilled water --is used as a general purpose enrichment medium for not particularly fastidious microorganisms

diaphragm

adjusts the amount of light stricking the specimen opens and closes bw the condenser lense and the specimen

What are Slant medias used for

agar slants are generally used to grow stock cultures that can be refridgerated after incubation and maintained for several weeks since they can be capped which helps keep the agar from drying out. good for a back up incase something is to go wrong with your other samples. has more nutrients per unit area so bacteria has more nutrients than in a plate

taxonomy

classification, descripiton, identification and naming of living organisms

Cellular arrangments of bacterial cells

coccus, bacillus, vibrio, coccobacillus, sprillium, spirochete, diplococcus, tetrad, streptococcus, staphlycoccus, streptobacillus • Caused by how many planes of division they have. Coccus with just one plane of division maks a diplococcus, were as if it had two planes of division it would form a tetrad

SPONTANEOUS GENERATION Lazzaro Spallanzani--> heated broth

controls: heated broth in sealed and unsealed jars with plant and animal matter in them results: heated sealed jars had no growth, but heated open jars did, and sealed jars that are opened allowed growth Conclusion: no spontaneous generation backlash: needed to be open to allow "life force" to grow

rheostat

dimmer switch that controls the intensity of the illuminator (light intensity knob)

Fastidious organisms

fastidious organism will only grow when specific nutrients are included in its diet. The more restrictive term fastidious microorganism is often used in the field of microbiology to describe microorganisms that will grow only if special nutrients are present in their culture medium.

how are phylogenies used for modern taxonomies?

groups of organisms are arranged by how closely related they are thought to be. In early phylogenetic trees, the relatedness of organisms was inferred by their visible similarities, such as the presence or absence of hair or the number of limbs. Now, the analysis is more complicated. Today, phylogenic analyses include genetic, biochemical, and embryological comparison

What is filiform growth

grow that is dense and opaque with a smooth edge (opposite is effuse)

illuminator

high intensity bulb below the stage to light up image at higher magnifications

SPONTANEOUS GENERATION Francesco Redi --> meat and maggots

hypothesis=preventing flies from having direct contact with the meat would prevent appearance of maggots controls: open jar, corked jar and guazed jar results: maggots developed in open jars but non on the guaze covered or corked jars conclusion: maggots can only form when flies are allowed to lay eggs in the meat and that the maggots are offspring of flies no spontansous generation

coarse focusing knob

large scale movements up and down to focus image (only with 10x ob.lens)

alpha hemolysis

leaves highly pigmented but no clearings on blood agar --many normal bacteria cause this

Bacterial exotoxins in blood agar

membrane damaging -- form pores in membrane or enzymatically damage membrane components

x-y mechancial sstage adjusstment knobs

move the stage in the x-y plane to position speciemen in right place

what do slants, plates and broth have in common?

need aseptic inoculation can all help in identifying a microbe

Liquid media (broths)

o Used to grow microbes when fresh cultures or large numbers of cells are required o Differential media broths can be used in microbial identification

Why is it necessary to have fresh cultures for Gram staining

older bacteria may have damage to their cell walls that causes them to appear gram- even if it is gram + • Some gram postitives loose their ability to retain the crystal violet-idone complex in as little as 24 hours of incubation, thus it is best to do gramstains on cultures no older than 24 hours old for the best results

condenser lens

on the stage. condense light from the illuminator so that all light rays on the specimen so that it is maximally illuminated

classification

practice of organizing organisms into different groups based on their shared characteristics

How do environmental factors select for microflora with particular physiological traits

• nature of the environment selects the predominant microflora with the appropriate physiology. Selective factors include availability of nutrients and energy sources, pH and temperature. this is why we see a differing communities in different parts of the digestive tract because each has different selective factors

explain the difference bw magnification and resolution

•MAGNIFICATION= How many times larger the specimen looks (new size divided by original size) RESOLUTION= how clear the image looks • How far apart two points must be for the microscope to view them as separate • Resolution improves as the limit of resolution gets smaller

Bright field microscopy

simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light and contrast in the sample is caused by absorbance of some of the transmitted light in dense areas of the sample • Compound microscope with one or more lenses that produce a dark image on a bright background.

simple stain

single dye is used to emphasize particular structures in a specimen/ makes it all the same color doesnt give much info besides size and morpholgy maybe

acid dye

the chromophore is a negative ion would stain positively charged molecules and structures such as proteins

basic dye

the chromophore is a positive ion would stain negativly charged structures such as nucleic acids and proteins

what is a positive stain

the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains. typically get absorbed by the cell or organism being observed adding color to objects of interest

Simple and complex media are similar in that...

they allow for bacterial growth

how are selective and enrichment media similar?

they are trying to promote the growth of a specific organism

what happens to light when magnified

they becomes dimmer because there is less light per unit area of image

PROTAZOA

-- backbone of many food webs -- provide nutrients to other organisms --have cillia and flagella --some photosynthetic some feed on organic material -- informal term for single-celled eukaryotic organisms, either free-living or parasitic, which feed on organic matter such as other microorganisms or organic tissues and debris.

FUNGI

-- eukaryotes -- multicellular (somemtimes) --yeasts, molds --can fix nitrogen for plants

gamma hemolysis

-- non hemolytic

Ocular lense

-- part of the eye piece --10X magnification

Flagella staining

-- thickens the flagella first via application of mordant (tannic acid) --this coats the flagella --stained with pararosaniline orbasic fushin

ARCHEA

-- unicellular/ prokaryotic --extermophiles --differences in genetics and metabolic pathways/cell walls

Plated media

typically used for obtaining isolation of species, differential testing (seeing if it is one thing or not), and quantifying bacaterial densities

what would happen to cells with cell walls when placed in hypertonic solution

water particle move out of the cell and the cell membrane shrinks and detaches from the cell wall PLASMOLYSIS

what would happen to cells with cell walls when placed in hypotonic solution

water particles move into the cell and the cell wall counteracts osmotic pressure to prevent swelling and lysis

what would happen to cells without cell walls when placed in hypotonic solution

water will move in and cause the cell to swell and eventually lyse

Differential staining

-- used to tell if and organism is something or not based on outcome of stain --Gram stain is most common (says if a bacteria is + or -) --allows determination of size, morphology, arrangement --example= acid fast stain, capsule stain (does it have one or not), spores, and flagella

VIRUSES

--Acellular (not cellular) --just have proteins and genetic material -- require a host to grow

ALGAE

--Eukaryotic --photosynthetic organisms that extract energy from the sun --release oxygen and carbohydrates into their environment

Acid fast stain

--For gram positive cells --some have waxy mycolic acid in their cell walls and some dont (so checking for that) --Ziehl-Neelsen technique and kinyoun technique --primary stain is carbofushin -- waxy acid fast cells retain the carbofushin even after decolorizing agent (acid alcohol) is applied --counterstain with methylene blue and stains the non-acid fast cells blue

How are microogranisms classified/distinguished as a unique species

--Leeuwenhoek used his simple microscope to observe single celled organisms, which he described as "animalcules" swimming in a drop of rain water --Pasteur showed that individual microbial strains had unique properties and demonstrated that fermentation is caused by microoganisms/ invented pasteurization to kill microbes that cause spoilage/made vaccines for the treatment of diseases -- Koch demonstrated connection bw single isolated microbe and human disease

why is a streak plate the first step in identifying an unknown organism

--because obtaining isolation of individual species from a mixed sample is generally the first step in identifying an organism --a bacterial sample (typically mixed) is streaked over the surface of a plated agar medium to get isolated colonies consisting of only the orginal cell type --getting it out there to make a pure culture

SPONTAENOUS GENERATION Pasteur-->swan neck flasks

--boilled broth to sterilize --due to swan neck, air was allowed inside, but microbes would be stuck in the kinks of the neck --allows "life force" to enter --but nothing grew, even though there was air flow --if swished the broth into the neck, then growth occured bc of contanct with microbes stuck in the neck so no spontaneous generation

beta- hemolysis

--distinctly clear zones and is caused by a battery of lytic enzymes that cause massive destruction of the blood cells --streptococcus (pathogenic strain) shows this --complete lysis of RBC

objective lense

--end of the body tube --on a rotating nose piece --typically 3 lenses of 10x 40x and 100x

Sabouraud's Agar

--has polypeptone, glucose, agar, di water and a pH of 5.6 --selects for molds and yeasts --preference fro acid pH and high sugar content, so is selective to these organisms

why is oil immersion lens necessary for high magnifications

--helps fill the space bw the oil immersion lens and the speciemen --oil has refractivve index similar to glass, so it increases the maximum angle at which light is leaving the speciment can strike the lense --this increase the light collected (NA) and betters the resolution

Blood agar

--produce lytic zones caused by secretion of enzymes that destroy the blood cells --degrade hemoglobin to greenish yellow bile pigments --alpha-hemolysis and beta hemolysis and gamma can show up, makes this differential media --rupture or lysis of red blood cells are indicative of bacterial exotoxins

BACTERIA

--prokaryotic --most are harmless, some pathogenic --wide range of metabolic capabilities in many environments using many nutrients --some photosyntheic and anoxgyenic --a member of a large group of unicellular microorganisms that have cell walls but lack organelles and an organized nucleus, including some that can cause disease.

why cant mitochondria and cholorplast multiply outside the cell

--they lost a good amount of genetic info so cannot replicate anymore so officially organells

PROTIST

--unicellular eukaryotes -- not plants, animals or fungi --(algae and protazoa are protists)

Endospore stain

--use heat to push primary stain (malachite green) into the endospore --washing with water decolorizes the cell, but the endospore retains the green stain --counterstain with safranin --resulting image reveals the shape and location of endospores if they are present --green endospores are seen in the pink vegatative cell

Capsule stain

--use negative staining technique to make halos around the border of the cell -- doenst need heat fixation --india ink is used to stain the background, leaving a clear area of the cell and the capsule

Endosymbiotic theory of mitochondria and chloroplasts

--• Based upon the chloroplasts' ability to reproduce independently, Russian botanist Konstantin Mereschkowski (1855-1921) suggested in 1905 that chloroplasts may have originated from ancestral photosynthetic bacteria living symbiotically inside a eukaryotic cell. He proposed a similar origin for the nucleus of plant cells. This was the first articulation of the endosymbiotic hypothesis, and would explain how eukaryotic cells evolved from ancestral bacteria --both have ribosomes that resemble prokaryotic ribosomes --both have own DNA that resembles proyarotic DNA --both reporduce by binary fission like prok

what would happen to cells without cell walls when placed in hypertonic solution

water will move out of the cell causing it to shrink, CRENATION

stage

where the glass slide with the specimen is clipped into place

HELMITHS

worms that are parasitic

why are genetic methods better for microbes than relying on observable characteristics

you can distinguish more than what is visibly seen. many microbes may have similar morpholgies, colors and other visible traits, but still be different in non-visible ways (metabolism and such)

Describe the steps for obtaining a pure culture from a mixed culture

• A mixed culture contains two or more species where as a pure culture only has one • Obtaining isolation of individual species from a mixed sample is generally the first step in identifying an organismnormally done through isolation technique via streak plate 1. Label the plate with name, date, and sample 2. Obtain mixed culture 3. Leave sterile agar plate on the table and lift the lid slightly, using it as a shield from airborne contamination 4. Drag loop back and forth across the agar surface 5. Remove loop and replace lid 6. Sterilize your loop via flame 7. Rotate 90 degrees 8. Let loop cool for a few moments then perform another streak 9. Do this for 4 streaks (quadrant style) 10. incubate can also use a spread to get growth of bacteria and then streak from there to isolate

Negative stain

• Absorbed by the background but not by the cells or organisms in the specimen to produce a silhouette of the organisms against a colorful background doesnt require heat fixing!!! negative chromophore in acid dyes are repelled by most cell walls and make a halo around the specimen

Koch's postulates

• All healthy organisms do not have causitative agent there • All diseased organisms should have this causitive agent (pathogen) • Agent must be isolated from diseased organism and grown in pure culture • Once you have one pure culture of suspected pathgen • Put in healthy animal, then die from that disease then get the same agent out of that organism • This proves germ theory of disease • He developed the pure culture methods (growth on solid media and isolation of pure cultures) • Used the scientifc methods to prove PROBLEMS Only 1 % of all microbes are able to be isolated Viruses cant really be isolated Differences in immune systems/susceptibility to disease There is a genetic basis for susceptibility to certain infections Causal agents are host-specific Some experiments would be unethical

what is the role of colony morphology in identification of different microbes

• All these morphological aspects are determined by the genetic makeup of the organism, thus can be very helpful in identifying the microbe

Steps for heat fixed slide for staining

1) Must let drop of water mixed with bacteria air dry before heat fixing (or else will boil and the bacteria will not adhere to the slide) 2)pass through the upper part of a flame two or three times (coagulates cytoplasmic proteins to make them more visible and heat fixing adheres the speciemen to the slide)

Gram Staining

1) Stain cells with crystal violet so that both gram neg and gram pos are stained pruple 2)iodine is used as a mordant so that the dye clumps up and enhances staining (makes crystal violet iodine complexes stuck in pep wall) 3) alcohol is used to decolorize by taking the crystal violet out of the gram negative since the peptidoglycan is smaller, but stays in the gram positive ( 4)gram negative is counteredstained with safranin so that you can tell the difference bw neg and pos

How can prokaryotes be found in nearly every habitat on earth?

• Bacteria can live all over your body and have prime environments to live either on our nose, our GI tract, or a hot spring. It all depends on metabolism and what envrionments that they can grow in, they are also good at adapting to different environments • They can live in or on our cells and there are more bacteria than human cells so they are everywhere

How would incubation conditions (medium, time, temp) affect colony's appearacne

• If bacteria is growing in medium that does not provide the right nutrients, it will not grow vs if given nutrients. It will grow and flourish more if the right nutrients are present • If not given adequate time to grow, you may not see many colonies or the full morphological appearance may not of had time to become present. Thus enough time must be given to see all the normal charcteristics • If not being grown in the correct temperature the bacteria may die off or not grow adequately. The bacteria needs to be in its optimum range of heat to grow maximally (pigment is stronger or weaker at different temps bc enzyme producing pigment may be funcational at certain temps)

Demonstrate the quadrant streak method for isolating pure colonies

ALWAYS FLAME LOOP AND LET COOL BEFORE STREAKING EACH QUADRANT

what is contrast

CONTRAST=the difference in light intensity bw the image and the adjacent background

What is CFU? why is more correct than colony?

Colony Forming units --more correct than colony since some colonies form from individual cells and others from pairs, chains or clusters of cells --colony by definition means that all cells have the same genetic material and are clones, but if two different colonies grew close and merged with each other that isnt necessaryily true

Enrichment media

Enrichment media=contain added essential nutrients a specific organism needs to grow (can allow for growth of other organism that need similar nutrients) (preferential growth of desired microorganism that represents a fraction of the organisms present in the incoculum) example = if we want to isolate bacteria that break down crude oil, hydrocarbonoclastic bacteria, sequential subculturing in a medium that supplies carbon only in the form of crude oil will enrich the cultures with oil-eating bacteria

GYC

Glucose-Yeast-Calcium Carbonate Agar (complex media) --rich, differential, and can select for things that use glucose --no fructose so no polysaccharide capsule --isolates bacteria that form acids (lactic acid) by glycolysis (glucose is fermented to lactic acid) --calcium carbonate neutralizes the acid and causes a clearing around the colony (insoluable in precipitate in agar) (lactic acid made solublizes=clearing) makes it differential

how did ignaz semmelweis obseve that mothers in wards staffed by midwives had lower mortality rates than in ward staffed by phsyicans and med students

He suspected that the student scarried disease from the autopsies to the patients they examined. His suspicions were supported by the untimely death of a friend, a physician who contracted a fatal wound infection after a postmortem examination of a woman who had died of a puerperal infection. The dead physician's wound had been caused by a scalpel used during the examination, and his subsequent illness and death closely paralleled that of the dead patient. Although Semmelweis did not know the true cause of puerperal fever, he proposed that physicians were somehow transferring the causative agent to their patients. He suggested that the number of puerperal fever cases could be reduced if physicians and medical students simply washed their hands with chlorinated lime water before and after examining every patient

Germ theory of disease evidence

Joseph Lister, developed a technique using phenol to disinfect surgical wounds 1. Denatures proteins so that they cannot fold/disrupts the cell membranes Ignaz Semmelweis 1. Determined that it was doctors that did not wash their hands after autopsies that infected mothers after childbirth to get sick and die from disease. Did not know what caused the disease, but washing hands reduced maternal deaths John Snow 1. Used epidemiology to trace the source of disease outbreak 2. Determined that it was contaminated water that caused transmission/outbreak of cholera in London 3. Used cholera death maps to track what water mill was contaminated and causing the most affected by the disease

what would happen if a step or steps in the gram stain prep were done wrong

OVER-DECOLORIZE • Leaving the decolorizer on too long and you will get reddish gram positive cells UNDER-DECOLORIZE • Produces purple gram negative cells • Stain crystal from an old or improperly made stain solution can disrupt the field or stain precipitate may be mistakenly identified as bacteria

Explain the difference bw a simple and compound microscope

SIMPLE MICROSCOPE • optical microscope , often referred to as light microscope. It is a type of microscope which uses visible light and a system of convex lens to magnify images of small samples. It gives you nothing but a clear and magnified image of your sample. a magnifying glass is like this COMPOUND MICROSCOPE • microscope fitted with two or more convex lenses. The high magnification produced by these lenses together enables a detailed study of micro-organisms, cells and tissues. • Has a built in light that it uses, not just natural light

Selective media

Selective media= favor the growth of some microorganisms (inhibit the growth of unwanted microogranisms and support growth of the organisms of interest by supplying nturients and reducing competition) example = Macconkey agarhas bile salts and crystal violet which interefere with growth of many gram + bacteria and favor growth of gram -

explain how you could improve the resolution of a high magnification image?

Shorten the wavelength of the light being used or increase the numerical aperature (ability to gather light) via oil immersion

fine focusing knob

Small scale up and down movements (should be used fo 40x and 100x)

what are the pros and cons of light microscopy with and without staining

Staining -pro=better contrast bw the speciemen and the background, so you can see more characteristics since they are usually transparent -con= this kills the cell no stain -pro= the organism lives -con= dont see as many details, since mostly cells are trasnparent

STYA

Sucrose-Yeast-Tryptone Agar (complex media) --helpful in detecting streptococcus salivarius from the large variety of microorganisms that are on a tounge plate -- allows S.mutans to produce exo polysaccharide capsules --tiny gummy colonies --enriched, differential, and selective