Flashcards in Lab Practical 1: Study Guide Deck (174)
____ means "without microorganisms"
______ refers to practices that help reduce the risk of microbial exposure, transmission, and contamination.
Aseptic techniques ____ or kill microorganisms from hands and objects, reducing the risk of transfer of potential pathogens.
Aseptic techniques employ ______ instruments and other items.
Aseptic techniques _____ patient's risk of exposure to microorganisms.
_____ are 100-1000 times smaller than most mammalian cells; they range from 0.5 to 3 micrometers.
Microorganisms differ widely in ____ and ____.
shape and size
The majority of bacteria are either spheres or ____.
shaped spheres are called ____.
cocci, plural coccus
shaped rods are called ____.
bacilli, plural bacillus
a mix between cocci and bacilli are called _____.
coccobacili, plural coccobacillus
curved rods are called ____.
The ____ and ____ of the cells with their staining properties are used for classifying and preliminarily identifying clinical isolates.
shape and arrangement
To grow and isolate microorganisms, the specimen is spread (______) onto agar media containing nutrients to yield individual colonies. These individual ______ can be then further tested for identification.
_____ is used to grow (culture) microorganisms.
Different types of media used in the lab are to be chosen to suit the ______ of the species to be grown.
Typically, media is a mixture of nutrients, moisture, amino acids, _____, buffers, etc.
Two main categories of media used for microbial growth
artificial culture media
tissue culture media
This type of media contains the necessary nutrients to grow a variety of bacteria and fungi.
artificial culture media
This type of media can be solid or broth form.
artificial culture media
______ media is broth that has had agar added to it allowing it to take on the consisenticy of firm gelatin.
artificial culture media
Certain bacterial species of clinical interest will not grow on artificial culture media, including ______ and ______ as well as viruses.
chlamydiae genus: Chlamydia
Rickettsiae genus: Rickettsia
This type of media contains living cell/tissues which act like "host" cells in order to grow and study viruses and other obligate intracellular organisms.
tissue culture media
These two obligate intracellular pathogens need tissue culture media to grow.
3 forms of artificial media
_____ is a liquid medium that becomes turbid as bacteria grow in numbers. There are many different types.
_____ refers to the change that occurs in broth as bacteria grow - the more bacteria grow, the more they increase the cloudiness of the broth, changing it from the original clear to "cloudy" appearance.
______ is agar, derived from seaweed, is added to liquid media, heated and allowed to solidify so that the agar surface in the test tube is slanted.
____ is agar poured into a petri dish and allowed to solifify. This produces a relatively large flat surface for bacterial growth and isolation.
3 varieties of agar and broth
_____ is media that supports the growth of most (but not all) bacteria of clinical interest.
3 types of non selective media
sheep's blood agar (SBA)
Trypticase soy agar (TSA)
nutrient agar (NA)
_____ is media that encourages the growth of one species or group of species while inhibiting the growth of others.
____ can be useful when attempting to grow a particular organism, while inhibiting the growth of other organisms which might be found in the same environment.
Selective media often contains certain ____ in low concentration, bile salts, high NaCl concentration or other substances to inhibit and/ or encourage growth.
_______ allows for a specific metabolic activity of an organism to be detected by visual inspection of the growth of the organism. This is often accomplished by observing changes in the ____ of a pH indicator.
______ can provide information about characteristics of various microbes aiding in their identification.
4 types of differential media
triple sugar iron agar (TSI)
mannitol salt agar (MSA)
sheep's blood agar (SBA)
Some media can be both differential and selective. They ____.
do not have to be both
What variety is Mannitol Salt Agar?
selective and differential
_______ contains 8.5% salt which inhibits many common microorganisms, while allowing for the growth of Staphylococcus species which are naturally salt tolerant.
Mannitol Salt Agar (MSA)
What variety is MacConkey Agar?
selective and differential
_____ is selective for gram negative orgnaisms. It contains bile salts and crystal violet dye which inhibit the growth of gram poisitive organisms.
_____ media is differntial for lactose fermentation. Lactose ferminting species turn dark purple/pink while lactose negative species remain tan or light colored.
______ is made by adding 5% sheep's blood to a nutrient TSA medium. The intact red blood cells give the medium a bright red color similar to the color of fresh blood.
Sheep's blood agar (SBA)
What variety is sheep's blood agar?
non selective and differential
Sheep's blood agar allows for the growth of a variety of microbes, but the media is differential by means of ____.
red blood cell hemolysis
____ hemolysis is the complete breakdown of RBCs. Bacterial cells secrete ______ hemolysins that completely lyse the RBCS.
The result of beta hemolysis results in a clearing of the "blood" around the colonies that can appear ____ to clear depending on the strength of hemolysis.
_____ hemolysis is the partial breakdown of RBC. Bacterial cells secrete hemolysins that cause lysis of the RBCs without completely breaking down the hemoglobin. This leaves a _____ hue in the medium.
______ hemolysis is no hemolysis. Bacteria grow on blood agar without disturbing the red blood cells -leaving them intact.
Lab 1 compared ways to reduce bacterial contamination by comparing ____ with ____.
Hand washing is a mechanical ______ of microorganisms that results from soap creating an emulsification of oils and water, trapping dirt and microbes in the process.
Alcohol based hand sanitizers contain a high enough concentration of alcohol (typically 60-95%) to have a ______ on most bacteria and viruses.
Unlike soap and water, hand santiizers do not remove ____. So it is recommended that people in health care settings who use hand sanitizer also use soap and water intermittently throughout the day.
dirt and grime
What was more effective at reducing bacterial numbers....soap or hand sanitizer?
____ is normal and permanently colonizes the haost and is established during the first few month of life.
______ is normal and colonizes the host for a short period of time and do not persist in the host's body due to the body's defenses.
Do we want our skin to be "sterile", completely free of microbes?
No, microbes that live on the skin prevent other dangerous microbes from being able to get a foothold. Microbes have also been show to prime the immune system to respond to pathogens.
______ is the ability to see two items that are close together as two separate and individual objects.
_______ is a numerical measure indicating the resolution of lens, the smaller the distance between objects that can be distinguished, the greater the _____ of the lens.
resolving power of lens
In a ____ microscope an object is in focus at low magnification it will remain in focus when switching to a higher magnification.
In a _____ microscope, when an object is centered in the field of view at low magnification it will remain centered when switching to a higher magnification.
____ is the fending of light as it passes from one medium to another of different density.
_______ is the measure of how greatly a substance slow the velocity of light (causing refracation)
______ indicates the magnification power of that lens alone.
____ is defined as the total magnification power of all lenses used. In this class we will be using a ____ light microscope which use use two lenses to magnify an image.
Ocular lens has a magnification power of ____.
There are 3 to 4 different ____ lenses you can choose from that range from 4 x, 10x, 40x, and 100x.
____ is the ocular magnification x the objective magnification.
___ is the lens that you look through.
ocular lens (10x)
______ aka turret is what holds the objective lenses and can be rotated to lock each objective into place directly above the specimen.
______ range in power from 4x to 100x. The shortest lens is the lowest power, the longest lens has the greatest magnification power.
____- is the flat platform where you place your slide.
____ hold teh slides firmly in place.
_____ is the smaller round knob that is inset into the course adjustment knob on the side of the microscope. This is used to fine-tune the focus of your specimen. This know also focuses by adjusting the distance between the specimen and the objective, but at much smaller increments that the course focus knob. Higher power lenses should use this knob only.
fine focus know
______ is the large round knob on the side of the microscope. IT is used for course or rough focusing of the specimen only. This serves to adjust the distance between the slide and the objective lens (either by moving the stage up and down, or moving the objectives). Should only be used with lower power lenses.
Coarse focus knob
_____ is used to focus the light that passes through the specimen.
_____ aka iris allows the user to adjust the size and intensity of the light beam that project through the slide from below. The proper amount of light your should let through will depend on the magnification used and the contrast of the specimen itself.
_____ provides a light source which illuminates the specimen from below. Most light sources allow the user to adjust intensity of light being emitted.
____ is the backbone of the microscope, providing support to the overall structure.
____ are located off the side of the stage. You will use these to move the slide and scan the specimen. One know moves the stage left and right, the other moves the stage forward and backward.
stage adjustment knobs
You should life the microscope using the ____ and base.
Lenses are only to be cleaned using the ___ with a few drops of lens cleaner. Use this to remove oil from lenses.
Use _____ to clean any oil off the stage and slides. Never use ___ on the lenses.
Oil has a similar refractive index as ____. By connectign the two refractive surface (____ and ____) the magnifications of 1000x can be reached while still preserving good resolution.
oil and glass
____ total magnification must be used for all bacterial specimens.
______ the smear is essential to good staining and visualization.
Heat fixing ____ the bacteria/sample to the slide.
Heat fixing ____ key enzymes in the bacterial cells, allowing them to pick up more stain and thus be easier to see.
Heat fixing will ____ a majority of the bacteria present.
How to heat fix?
1. take your dry smear prepped slide to the Bunsen burner
2. grasp the edge of your dry slide, pass your slide (speciment side up) through the flame of the bunsen burner three times
_____ uses only one stain (dye) to make the cells visible under the microscope.
Bacteria are ____, so to see them, and better see any epithelial cells than may be present from our gum line, we must stain them.
Gram-negative, spirochete (cork screw)
Gram-negative, diplococci - coffee bean shaped
Gram-negative, rod shaped, endospores (look like bicolored capsules)
Gram-positive, rod shaped ( a little curved)
Why do you want to let more light in when your go from a low magnification objective to a higher magnification objective?
the area in the field of view gets smaller as well as darker. To see the specimens clearly, additional light may need to pass through the slide.
Whey do we need to use oil with the oil immersion lens?
Oil has a similar refractive index as glass. By connecting the two refractive surfaces (oil and glass) the magnification of 1000x can be achieved while still preserving good resolution.
Aseptic technique employs laboratory methods designed to prevent ____ by undesirable organisms.
Aseptic technique protects your experiment from becoming ____, but it also protect you and the environment from becoming contaminated as well.
An ______ is a tool often used to inoculate different media in the lab.
The _____ can be used to effectively and precisely spread bacteria across teh surface of an agar plate.
When working in a lab, you must sterilize your inoculating loop to ensure that no ____ from previous experiments, or microbes from the environment, contaminate your experiment.
The incinerator must be preheated for ____ minutes prior to use.
When using an ____ place the inoculating loop in until the wire portion of the loop glows oragne-hot. Hold the loop at all times, do not "rest" the loop in the ____ or it will melt.
Cooling the loop ensures that you do not overheat a sample and create potentially dangerous _____.
Cooling the loop can be done two ways. Hold the loop in the air for ___ seconds. or touch the loop to a portion of the plate without ______ along the edge.
10 to 15 seconds
When using an inoculating loop to transfer bacteria to an agar plate, open the lid ___ degrees.
The ____ technique depends on the spatial isolation of a single bacterial cell on a solid medium.
streak for isolation (SFI)
We cannot see individual bacterial cells with the naked eye, but we can see the ___ that result after an overnight incubation.
A colony is formed from reproduction of a ______ cell so that all the members of a colony are descendants from that original cell.
The term _____ refers to the one bacterial cell that started the colony.
CFU or colony forming unit
During the streak plate technique you will be spreading our bacterial sample across the surface of an agar with the goal of achieving ____.
spatial separation of individual cells
A properly executed streak plate will give nice isolation of colonies, allowing for ________ of colonies that form after an overnight incubation.
If the streak plate technique shows colonies that are visually distinct (different), the original sample is a _____ sample.
mixed - contains more than one type of bacteria
If the streak plate technique shows colonies that are visually the same shape, size, and color the original sample was a ____.
pure specimen - one bacterial specimen
A streak plate is an excellent way to separate out bacterial species that are in a ____.
If a patient has a UTI, the streak for isolation allows you to choose and eventually test ____ colonies to see which potential pathogens you are dealing with.
What needs to be written on all agar plates.
Name - section - date - sample
Practice quadrants for SFI
Why do we incubate plates upside down?
Excessive moisture from the condensation of water, derived from the initial cooling of the hot sterile media, can collect on the inside of the lid and sides of the plate. If the water drops down onto the agar surface, spreading and mergin of colonies can occur. Always incubate paltes upside down.
If the inoculating loop is not sterilized between quadrants during the SFI the result will be ____.
equally heavy growth in all four sections of the plate
Properly executed streak for isolation technique showing successful isolation of individual colonies in the ___.
3rd and 4th quadrants
A ____ is a visible mass of microorganisms all originating from a single cell.
The ____ is almost always the first step in the identification of a bacterial organism.
The Gram staining method is used to differentiate bacterial species into ____ large groups; _____ and _____.
The difference between Gram-positive and Gram-negative is based on their ____ composition, specifically the levels of _____.
Gram positive bacteria is ____ in color.
Gram negative bacteria ____ in color.
Gram positive bacteria has a ____ of peptidoglycan.
Gram negative bacteria has a _____ of peptidoglycan.
Gram positive streptococci
Gram negative bacillus
Gram positive bacillus
Gram positive staphylococci
_____ can be made from broth cultures or from an agar plate.
Gram stain protoco
How to label a slide
gram positive, rods, streptococci
gram positive, cluster of coccous - staphylococci
gram-negative, rods, bacillus
Can a gram stain be used to as the only means to identify bacterial pathogens in a patient fecal sample?
No, a fecal sample will likely display dozens of bacteria within the sample. It would be a good first step, but idetifiying the particular pathogen by just gram staining would not be possible.
What is the difference between a simple stain and a differential stain?
a simple stain requires only a single dye. A differential stain requires a primary stain and a counterstain
Step 1 gram staining
flood slide with crystal violet (CV)
Step 1 of the gram staining is called the ____ that will enter teh cell wall and stain all the bacteria.
Step 2 of gram staining
Flood slide with grams iodine also called the mordant
The ____ will bind with the CV in the cell wall forming a large CV-I complex.
Step 3 gram staining
Drizzle slide with alcohol (decolorizer)
_____ dissolves the outer membrane lipids allowing the CV-I complex to escape the thin peptidoglycan layer of gram negative cells. Gram negative cells now colorless, gram positive cells still purple.
Step 4 of gram staining
Flood slide with safranin
Safranin is the ____ that stains everything pink but you will only see this in the gram negative cells because they were just streipped of all color by the alcohol.
After gram staining blot the slide gently with ____ paper to dry excess water.
______ is an effective way in which to control microbial growth.
In the healthcare industry _________ is used to sterilize open surfaces like in hospital rooms or operating room surfaces.
_____ is also used in some restaurants to keep food surfaces sterile and in lab biosafety hoods to control microbial contamination.
UV light controls bacterial growth by causing irreparable levels of damage to the ______.
bacterial cell DNA
UV light is a form of ____ radiation that creates thymine thymine dimers which inturn disrupts ____. This type of mutation is lethal to bacteria.
UV light is most effective when the light waves can come ____ with the surface.
in direct contact
Any substance like the plastic lid of a Petri dish can ____ UV rays will prevent the germicidal effects of UV.
UV light experiment examined the ___ and ____.
length of time of exposure
type of bacteria
Did one organism survive exposure to UV better than the other? Which one? What about this organism made it more resitant?
Yes, Escheriachia coli was more resistant to the UV light. The gram negative specimen has the thick outter membrane to sheild from the UV light.
What happened when the index card was not used?
more uv light would effect the bacterial cells even if indirectly ner the lgith
What would happen if the Petri dish lid was left on?
There would have been 100% growth in all sections of the Petri dishes since the lid blocked the UV light from having an effect on the bacteria.
What effect would endospore formation have on this experiment?
There would have been a greater number of bacteria left after UV light exposure if there had been endospor formation. Endospores are refractile so the UV lgiht cannot penetrate them to do damage to teh DNA, resulting in cell death.
The purpose of the Kirby Bauer Disk Diffusion Assay is to test ____.
The _________ is used to determine the susceptibility of bacteria to various antibiotics.
Kirby Bauer Disk Diffusion Assay