micro lab midterm

what kind of stain is a gram stain?

a differential stain

Aseptic technique refers to what?

a procedure performed under sterile conditions.

give two examples of margin shape

smooth entire lobate

An endospore's many layers protect the necessary molecules of life for the sporulating organism but also makes an endospore difficult to stain. _______ is used to force the primary stain ___________ into the endospores. Vegetative cells are also stained green but after rinsing with _______, the vegetative cells lose the primary stain and need to be counterstained with ________ to be seen as ________ cells among the green endospores.

- Heat -malachite green -water -safranin -red

name 2 acid fast bacteria

- Mycobacterium smegmatis ** maybe not important ---M. Tuberculosis

describe the procedure for making a smear from solid media

- Using a wax marking pencil, make a ½″ circle on a clean microscope slide. The smear will be placed within the circle. Write the initials of the organism to be stained on one end of the slide. - Flame the loop. If the smear will be directly stained, use the sterile loop to obtain 1 drop of water (the water can come from the tap) and place the drop on the slide within the circle. For indirect staining, place a drop or 2-3 loopfuls of primary stain like Congo red on the slide within the circle. - Flame the loop; allow it to cool for several seconds. Aseptically obtain a small amount of organism from the solid medium (a plate or slant). Lightly touch the cooled loop to the culture or you will get too many cells. If a slant culture is used, the tube must be flamed as shown in Laboratory Technique 2: Aseptic Technique, Figure 2T-1, illustration 7b. - Mix the cells on the loop into the drop of water (or primary stain for indirect staining) as shown in the figure. The water-cell suspension should be a pale film which is barely visible. (It should be almost clear, or it is too thick.) If the smear is too thick, simply add another drop of water and spread the smear over a larger area of the slide. Thickness of smear is difficult to judge for smears made in a primary stain for indirect staining. Flame the loop and set it aside. If the smear will be indirectly stained, let the smear air dry then proceed with the staining procedure (see Laboratory Technique 4). Do not heat fix or rinse smears that are indirectly stained. Continue with step 5 below if the smear will be direct stained. - Place slide with wet smear on slide warmer. Be sure slide is labeled with your name or initials, as many students are sharing the slide warmer. - Allow your slide to stay on the slide warmer for at least 5 minutes at 55-60°C. It is important to leave the slide on for a full 5 minutes, even if your smear dries before then. The additional time will heat-fix your smear to the slide so it will not wash off during staining. It is also important to wait until the slide warmer has reached at least 55°C. A lower setting may dry the smear, but not heat-fix it. (This is important mainly for the first section of the day when the slide warmer is turned on.) But be sure that the slide 11 warmer is not set above 60°C (setting 8 on the dial). Too much heat will cause cells to lyse and destroy cellular morphology. - After 5 minutes, remove your slide and place it on the staining rack in the stain trough. When the slide has cooled, it is ready to be stained. Be sure to rinse gently while staining. Although your smear should be heat-fixed, too aggressive rinsing can remove your specimen. - Alternatively, if slide warmers are not available, allow the smear to air dry by placing the slide on the bench top for several minutes. Do not dry the smear over the flame, as this may create an aerosol and cause the cells to rupture. When the smear has air dried, heat fix it by passing the slide over the flame back and forth. The glass heats quickly, so you may want to hold the slide with a clothespin. Do not hold the slide in the flame; excessive heat will damage the cells. If the slide is too hot to touch against the back of your hand, it was held in the flame for too long. When the slide has cooled, it is ready to be stained.

Gram reaction is most consistent on young, growing bacteria that are less than _________ old. Gram stain results may not be accurate for older cultures because many Grampositive cells begin to lose the ability to retain the primary stain and appear _______

24 hours Gram-negative.

A _________ is one containing a single kind of organism. Rarely do pure cultures exist in the natural world, but pure cultures are useful for lab scientists to study cellular processes without influence from other organisms. For example, pure cultures of Escherichia coli were used to determine the roles of DNA polymerases involved in replication and DNA repair.

-pure culture

Cells in biofilms communicate with each other through ________. This is usually mediated through a compound called _________. Cells in a biofilm produce this compound and when it has accumulated to a certain level, it causes effects in neighboring cells. These effects may be to recruit other microbial cells to join the biofilm or to induce all members of a biofilm to produce an enzyme or virulence factor at the same time. This simultaneous release of an enzyme by a large population of bacteria is more effective than a slow constant release by only a few bacterial cells.

-quorum sensing -homoserine lactone

describe the procedure for making a smear from a broth culture:

- Using a wax marking pencil, make a ½″ circle on a clean microscope slide. The smear will be placed within the circle. Write the initials of the organism to be stained on one end of the slide. - Obtain the tube with the broth culture. You will use your inoculating loop to transfer this culture to the slide, but it must be done aseptically (refer to Laboratory Technique 2: Aseptic Technique, Figure 2T-1). -After the loop has cooled, you are ready to go into the broth tube. This must also be done aseptically. Remove the cap of the tube with the ring and little fingers of the hand holding the loop. Pass the lip of the open tube through the flame. Always hold the tube at an angle to prevent contaminants from the air from entering the tube. Remove a loopful of broth culture. Pass the lip of the tube through the flame again, then replace the lid on the broth culture. -Place the loopful of the organism on the top side of the microscope slide within the circle and spread the contents in a thin layer that should dry almost immediately. If the smear will be indirectly stained, the loopful of culture will be mixed with the primary stain added to the slide first. Flame the loop again. A second loopful of broth may be necessary for dilute cultures. Continue with step 5 below.

In nature, microorganisms are/are not found in pure cultures and are usually not suspended in broth. Rather, they exist in microbial communities called________, which can be found at _________. We are probably most familiar with the biofilm that forms on our teeth between brushings. Other examples of biofilms include those formed on artificial joints, catheters, retainers, and contact lenses. There are other examples of biofilms in your home including the film that forms on pet water bowls, in aquariums, in your shower, and in dirty pots soaking in the sink. Examples of biofilms in the environment include the slimy layer on submerged rocks in a lake or the film on the surface of a stagnant pond.

- are not - biofilms -water-air or water-solid interfaces

Microorganisms must be viewed under magnification because they are too small to be seen by the unaided eye. In this class, one type of light microscope, a____________, will be used to observe microorganisms. Specific lenses magnify the image of a cell such that details of its structure are apparent. The microscope you will be using is called a __________ because it consists of a series of_______ and _________ lenses. The objective lenses are on a revolving nosepiece above the stage of the microscope (where a smear or wet mount to be observed is placed).

- bright-field microscope -compound microscope - ocular -objective

cocci -Cocci are round, and the cocci may remain in pairs called________after cell division. -If the cocci remain in chains, they are termed________ -Groups of four cocci are called _________, and grapelike clusters are called ________ Spiral bacteria have one or more twists. Vibrios look like curved rods, and spirilla have a helical shape that is rigid. Spirochetes are helical, flexible, and resemble a corkscrew. Some more complex morphologies include: Prosthecate cells—organisms such as Caulobacter crescentus and Hyphomonas neptunium produce prosthecae (sing. prostheca), cellular appendages that increase cell surface area and hence help transport of nutrients into the cell. Filamentous growth—some actinomycete bacteria, e.g., Streptomyces coelicolor, produce filaments or hyphae that form a mycelium(network of hyphae). The hyphae later differentiate and fragment to produce spores.

- diplococci - streptococci. -tetrads -staphylococci.

what are some characteristics used to describe cell appearance:

- whole colony shape - margin shape - elevation - optical properties - surface characteristics - pigmentation

what is an endospore? Where is it commonly found?

-An endospore is a specialized dormant structure that is commonly found in certain Grampositive rods such as those of the genera Clostridium and Bacillus.

-what is an example of a pure culture? -Can broth cultures be pure cultures?

-An isolated colony on an agar plate -yes, if they only contain one type of organism

what are examples of bacteria with capsules

-Azobacter Vinelandii (polysaccharide capsules) -Bacillus Anthracis (polypeptide capsules) -Streptococcus mutans -Streptococcus pneumoniae

name gram positive, rod shaped cells with endospores

-Clostridium botulinum -C. sporogenes -Bacillus stearothermophilus - Bacillus Sphaericus - B. megaterium - B. subtilis

Today, you will view capsules using Maneval's capsule stain, which uses Congo red and Maneval's stain. The acidic stain, ________ , stains the background, while the basic stain, ________, stains the cells. When viewed under the microscope, the background appears ________, the actual cells are ________ while the capsules remain and appear as a clear area surrounding the cell

-Congo red -Maneval's stain - red to reddish brown -unstained

A smear can be made from cultures in liquid or on solid medium and the procedure varies slightly with the culture medium used and with the direct or indirect stain procedure. -How are direct stains made? - How are indirect stains made?

-For direct stains, a slide is labeled with the culture to be stained, the smear made (description follows), dried, and heat-fixed using the slide warmer. - For indirect stains, the smear is made in the primary stain and the smear is never rinsed in the staining procedure, so heat fixation is not required.

-Why is immersion oil used? -Only the ________ oil immersion objective lens on the lab microscopes is designed for use with immersion oil.

-Some light from the condenser is refracted and does not enter the lens. Immersion oil, which has the same refractive index as the glass slide and lens, is placed between the specimen and an oil immersion lens. The use of this oil reduces the amount of light lost and increases resolution -100×

name three gram negative bacteria

-Spirosoma linguale (gram negative) - Escherichia coli -Azotobacter vinelandii

-Describe the bacteria in the genus Mycobacterium: -Mycolic acid is a group of complex branched-chain hydroxy lipids complexed to the peptidoglycan of the mycobacterial cell wall (Figure 4T-H). This lipid component renders these cells difficult to stain by traditional procedures such as the Gram stain. Several species of the genus Mycobacterium are found in the soil; others are important pathogens. Robert Koch discovered that gentle heat is useful for driving stain into Mycobacterium cells. Once stained, however, the cells resist rigorous decolorization, even using acid-alcohol, a more potent decolorizer than ethanol by itself or water. - Why are cells of the Mycobacterium species called acid fast?

-They are gram positive, non-endospore forming, nonmotile, rodshaped bacteria that possess unique lipid components called mycolic acids -Because they resist decolorization

What advantages do solid media offer for the culture of microorganisms compared to liquid media?

-You can determine colony morphology that could help you identify the microorganism -You can obtain a pure culture.

-A culture medium can be prepared as a liquid, which is called a _____, or as a _______, which contains a solidifying agent (usually ________). Agar is extracted from Rhodophyta, red algae, and is useful as a solidifying agent because after being heated to 100°C it will solidify at 40-42°C and will not melt again until the temperature reaches 80-90°C. Solid media in Petri dishes are called _______. The surface of plates can be used for the isolation and growth of microorganisms. - What are deeps? - What are slants?

-broth -solid medium -agar -plates -Deeps: test tubes containing solid medium, which has been allowed to solidify in an upright position, and are usually used for anaerobic growth. Slants: test tubes containing solid medium that has been allowed to solidify at an angle and the surface of the agar is used for the maintenance of stock cultures of microorganisms.

In the Ziehl-Neelson Acid-fast Stain, _________ (a magenta stain) is the primary stain used, and is a mixture of the dyes basic fuchsin and phenol. Phenol and heating enhance penetration of the fuchsin into the lipid of the cell. After decolorization with ________ (3% HCl in ethanol), the counterstain __________ is added. Cells that are not acid-fast have lost the primary stain during decolorization and will take on the counterstain and appear ________

-carbol-fuchsin -acidalcohol - methylene blue - blue

Microorganisms growing on a solid culture medium (e.g., agar plate) will form _________ if the cells used to inoculate the plate are separated from each other on the surface of the medium. A single cell will replicate and give rise to a single colony. Some of the characteristics of these colonies, such as the shape of the colony, the form of the colony margin, and the elevation and pigmentation of the colony, are visible to the naked eye. These properties, collectively referred to as __________, often differ between microorganisms, and hence provide useful information for description of the organism. Note however that genetically different organisms can share the same colony morphology. It is important to be able to recognize the colony morphology of microorganisms with which you are working in the lab. If colony morphology changes over time, the organism under investigation may also have undergone change. If more than one type of colony is present on a plate, this may indicate the presence of a _________, an unwanted organism which has been accidentally introduced into the culture. The colony morphology of a microorganism is dependent on the ________(pl. media) on which it is growing. The type and amount of nutrients present in the medium will influence the ______, ______, and even the ______ of a colony.

-colonies -colony morphology -contaminant -medium -size -shape -color

Capsules may have several functions in bacteria in addition to _______. They are important for ___________ of certain pathogenic microorganisms to host cells. An example of this is found in the oral bacterium, Streptococcus mutans, which produces dextran and levan which are used by the bacteria to adhere to tooth enamel. This tight adherence to the tooth surface is required for the development of dental caries (tooth decay). Encapsulated bacteria may also resist being engulfed by the phagocytic cells of the host's immune system. In the lungs, Streptococcus pneumoniae produces a capsule which resists phagocytosis and enables the organism to cause pneumonia. Encapsulated strains may be very invasive. If a capsule enhances the organism's ability to cause disease, it is considered a ________. Two variants of the same species may exist: one that forms a capsule and may be a virulent pathogen, and a nonencapsulated form which does not cause disease and is avirulent.

-desiccation resistance -attachment -virulence factor

-A simple stain is a _________ that uses an aqueous or alcohol solution of a single basic dye. Direct staining colors the entire microorganism so that cellular shapes and structures are visible. - A __________ is an indirect stain that uses an acidic dye to color the background, leaving the cells colorless. Both direct and indirect stains allow you to see cellular morphology. Some cell surface structures such as capsules and flagella can also become visible with staining.

-direct stain -negative stain

To protect against contamination by unwanted organisms, it is necessary to sterilize media and any objects with which it will come into contact. Sterilization is the___________. A common laboratory method of sterilization is _______. The autoclave is a sealed device that allows the entrance of steam under pressure. Moist heat facilitates killing of all microorganisms, including bacterial endospores, which are extremely heat-resistant. The autoclave is heated to 121°C at 15 pounds per square inch (psi) pressure for 15 minutes-1 hour depending on what is being sterilized and how much. Solid media takes longer to reach 121°C than liquid media. Likewise, larger volumes take longer to reach 121°C than smaller 3 volumes. This method is frequently employed to sterilize liquid media and other items that are heat-resistant. Dry heat sterilization kills by oxidation effects. One of the simplest methods of dry heat sterilization is direct flaming. This procedure will be used many times this semester when inoculating loops and inoculating needles are held in the flame of a Bunsen burner until glowing red hot. It is important to flame the entire wire loop or needle to minimize contamination. When using hot-air sterilization, items to be sterilized like glassware are placed in an oven and heated to 170°C for 2 hours.

-killing or removal of all living organisms and their viruses from a growth medium -autoclaving

-Heat fixation does what? - Why must cells be adhered to the slide?

-kills the cells, destroying autolytic enzymes, and helps the cells to adhere to the slide, though most cells have proteins and sugars on their cell surface, making them naturally sticky. - Adhesion of the cells to the slide is essential or the cells will be washed from the slide during staining.

-Bacteria are differentiated by many factors, including morphology or shape, chemical composition, biochemical activities, and other factors. Staining allows the _________ of a cell to be determined. - The basic shapes of most bacteria you will encounter in this course are what?

-morphology -coccus (pl., cocci) - rod/ bacillus (pl., bacilli) - spiral. Cells are arranged: -as pairs (diplo) -in chains (strepto) -in clusters (staphylo) -or randomly.

Most bacteria have cell walls that contain _________, which consists of repeating disaccharides attached by a polypeptide to form a lattice. This lattice surrounds and protects the cell. Grampositive cell walls have _________ which make the structure rigid. In addition to the thick peptidoglycan layer, most Gram-positive bacteria contain ________. In contrast, Gram-negative bacterial cell walls consist of _________ surrounded by an outer membrane. The outer membrane consists of lipopolysaccharides (LPS), lipoproteins, and phospholipids. Gram-negative cell walls do not contain ________

-peptidoglycan -many layers of peptidoglycan -teichoic acids. (Teichoic acids are primarily an alcohol and phosphate and provide much of the wall's antigenic specificity.) -few layers of peptidoglycan -teichoic acids.

A capsule consists of layers of ________ or _________ which surround the ________ of bacteria. Azotobacter vinelandii is one of many bacteria which produce a polysaccharide capsule, while Bacillus anthracis produces a polypeptide capsule. Capsules will be formed only by capable bacteria in the presence of ________. The capsule may also be considered a _______ structure since it serves as a ________. If a bacterium does not have the genetic capability, it cannot form a capsule under any conditions. Water is the principle component of the capsule and may prevent desiccation of the cell.

-polysaccharide -polypeptide-containing material -cell wall -excess nutrients -survival -reserve energy store

Microorganisms present in water samples attach to surfaces and grow. They produce ________ that help trap nutrients and aid in the attachment of the microorganisms to the surface. This network of microbial cells in their polysaccharide matrix also provides protection for the inhabitants. A biofilm infection lining the inside of a urinary catheter, for example, is difficult to treat because antibiotics and immune cells cannot penetrate the layers of a biofilm. Biofilms can also cause trouble in industry as companies may spend millions of dollars trying to control biofilm growth in pipes, on boats, and on oil rigs.

-polysaccharides

-Media can be _______, ________, _______, or _________. - What is a selective media? - What is a differential medium? - Carbohydrate fermentation or enzyme production could be demonstrated on differential media. Phenyl ethyl alcohol agar (PEA) agar is selective only. It selects for Gram-positive organisms but tells nothing of the physiology of the organism growing on it. Mannitol salt agar (MSA) is both selective and differential. It selects for the growth of salt-tolerant organisms and differentiates mannitol fermenters (turn agar from red to yellow) from non-mannitol fermenters (don't change color of agar). See Experiment 10C for the use of MSA. Eosin methylene blue agar (EMB) agar is both selective and differential. It selects for Gram-negative bacteria and differentiates lactose fermenters from non-lactose fermenters. See Experiment 11: Detection and Determination of Coliforms for the use of EMB.

-selective -differential -both -neither -selective medium; a nutrient medium designed to favor the growth of certain microbes and to inhibit undesirable competitors. -differential medium; medium which provides a visible indication of a physiological characteristic of a microorganism.

Rod shaped (Bacillus) -Bacilli are rod-shaped cells that divide only across the ______, resulting in either _________, or ___________. -Coccobacilli are short oval cells that resemble cocci. This coccobacillus shape can be induced by less than ideal environmental growth conditions, or simply by the genetic makeup of the cell.

-short axis -diplobacilli (if they are in pairs) -streptobacilli (if they are in chains).

If a mixture of organisms is spread on a _______ surface, individual cells may be distributed on the surface so that each will grow to form a visible colony. A ________ is a population of cells which arise from a single cell growing on a solid medium. All of the cells in the colony should be genetically identical but for the few, if any, in which a mutation may have occurred. An _________ is an example of a pure culture. Broth cultures can also be pure cultures if only one type of microorganism is present. A broth culture appears cloudy when growth has occurred, but unlike examining growth on a streak plate, it is impossible to assess if that broth culture is a pure or mixed culture just by viewing the tube.

-solid nutrient -colony -isolated colony

Following activation, often ___________, _______, then _______ occur to return an endospore to a vegetative cell state. Germination is a rapid process in which endospore characteristics that are no longer needed, including resistance to heat and chemicals, are lost. Visible swelling due to water uptake also occurs during germination. ________ involves the synthesis of DNA, RNA, and bacterial proteins. The cell will emerge from the spore coat, and is again termed a vegetative cell.

-sublethal heat, germination, then outgrowth - Outgrowth

Acid fast stain: -Before performing this stain, look at the plate containing Mycobacterium. Notice on this solid medium that the colonies take on a compact, wrinkled appearance. This is due to ____________. Isolation of "rapidly growing" mycobacteria requires incubation for _______ or more, while those termed "slow-growing," including M. tuberculosis, can require three to eight weeks of incubation to produce observable colonies. - Why is egg albumin used?

-the cell wall's hydrophobic nature (note that the mycolic acid in the cell wall is the hydrophobic part), which may also render the cell strongly impermeable to nutrients and may partially explain the typical slow growth seen in Mycobacterium species. -three days - The presence of the surface lipids in Mycobacterium spp. also causes the cells to form tight aggregates that are hard to disperse in aqueous solutions. For this reason, egg albumin, which has high protein content, is used to make the smear for the acid-fast stain.

-Most of the ______ and _______ will be eliminated when sporulation is complete. No _______ will take place in the fully formed endospore. The endospore contains DNA, RNA, ribosomes, enzymes, and a few other important small molecules like small acidsoluble proteins (SASPs). _________ is found in all endospores and is located in the core. Endospores also contain high levels of calcium complexed with the dipicolinic acid. This complex may play a role in 23 protecting the spore DNA from damage. -Endospores can survive _______,________, and__________

-water - Cytoplasm -metabolic reactions (remember, cryptobiotic state) -Dipicolinic acid -extreme heat -lack of water -exposure to many toxic chemicals and radiation.

steps for acid fast stain

1 A) Prepare your smear: Add a small amount of egg albumin solution to a clean slide. Using your loop, place a small amount of Mycobacterium smegmatis from the plate labeled Myco and mix well with the egg albumin to disperse the cells. Add 1-2 loopfuls of E. coli to the mixture on the slide and stir the mixture using a loop. Attach a clothespin to the slide and allow smear to dry using slide warmer. B) Leaving the slide on the slide warmer, place a paper towel square on the smear. C) Cover the paper towel square with carbolfuchsin and let it sit on the slide warmer for one minute. 2. A) Remove slide from slide warmer with the clothespin still attached; place the slide on the staining rack above the staining trough and allow the slide to cool. Note: Once the slide is cool, remove the paper towel square with forceps and discard the paper towel square in the trash can. Do not put paper in staining trough. B) Rinse with water until the carbolfuchsin dye no longer flows from the smear. C) Decolorize with acid-alcohol until the slide remains only slightly pink. (About 10- 30 seconds.) 3 A) Rinse briefly with water and counterstain with methylene blue for 2 minutes. A) Rinse slide with water briefly, blot dry, and observe with the microscope under 10×, then 100× oil immersion. Use an alcohol wipe to remove oil from the 100× objective when you're finished.

gram stain procedure

1. A) Make a thin smear of culture on a microscope slide. Let air dry and heat fix or use a slidewarmer to dry and fix the smear to the slide. B) Cover the smear with the primary stain crystal violet. Leave stain on the smear for 1 minute. 2. A) Rinse the slide gently with water to remove excess crystal violet. B) Cover the smear with Gram's iodine and leave for 1 minute. 3. A) Rinse the slide gently with water to remove the iodine. B) Add ethanol dropwise onto the smear. You can let it sit on the smear for 2-3 seconds or let the ethanol run over the smear and off the slide as you hold the slide over the stain trough until crystal violet no longer runs from the smear. 4. A) Rinse the slide gently with water and cover the smear with the counterstain safranin. Leave stain on the smear for 1 minute. B) Rinse the slide gently with water and blot the slide dry with bibulous paper. C) Observe with the microscope under 10×, then 100× oil immersion. Use an alcohol wipe to remove the oil from the 100× objective when you're finished.

How many flagella does Chlamydomonas reinhardtii have?

2

What is a vegetative cell?

A vegetative cell is one that can grow and divide under optimal conditions. Activities needed to sustain the cell are occurring within the vegetative cell.

Which of following are good sources for obtaining a water sample to create biofilms in lab? a. lake b. pond c. ditch d. aquarium

All of them

When making a simple stain, which of the following steps will you do? Flood the smear with a single dye. Make a thin smear on a glass slide Rinse gently with water Blot dry with bibulous paper Heat fix the sample on the slide

All of them. Make a thin smear on a glass slide, Heat fix the sample on the slide, Flood the smear with a single dye., Rinse gently with water, Blot dry with bibulous paper

A doctor going into surgery vigorously washes his hands well and then suits up with gloves. Why might he still need gloves even if he washed his hands really well, even with antibiotic soap? a. Normal flora may be harmful when introduced into tissues or the bloodstream. b. The patient has their own transient flora. c. Washing hands do not rid them entirely of bacteria. d. More of doctor's normal flora has been exposed.

All. More of doctor's normal flora has been exposed., The patient has their own transient flora., Normal flora may be harmful when introduced into tissues or the bloodstream., Washing hands do not rid them entirely of bacteria.

Which of the following are in accordance with aseptic technique? (Choose the best answer.) a. Keep only necessary materials on the bench top. b. Pass mouth of tube through flame rather than holding the tube in the flame. c. Hold the culture tube top in the same hand with which you hold the loop. d. Always wash the bench top when entering the lab.

Always wash the bench top when entering the lab., Keep only necessary materials on the bench top., Pass mouth of tube through flame rather than holding the tube in the flame., Hold the culture tube top in the same hand with which you hold the loop.

When will an endospore form?

An endospore will form within a genetically capable cell when essential nutrients are depleted or when water is unavailable. They are highly durable dehydrated structures.

The steps in biofilm production are... (select all that apply) Select one or more: succession Development Attachment colonization enrichment

Attachment, colonization, Development

Endospores may be located in a _______________ position. Select one: a. subterminal b. external c. terminated d. subteranean

The correct answer is: subterminal. They can be subterminal, central, terminal, and swollen sporanglum

How will you secure the coverslip to the depression slide for the hanging drop method?

The correct answer is: with petroleum jelly

Microscopes you will be using in lab to view your smears are: Select one or more: a. Compound light microscopes b. Single ocular microscopes c. Equipped with 1000x objectives

Compound light microscopes

what is the decolorizer for the acid fast stain? What does it do? Select one:

Decolorization with acid alcohol removes the carbolfuchsin from the non-acid-fast cells.

what will be counterstained in acid fast stain

E. coli

While performing the acid-fast stain you accidentally forgot to add the methylene blue. The acid-fast bacteria will appear colorless. a. False b. True

False. They will appear red/pink

Which of the following will be done to the biofilms? a. Gram stain b. acid-fast stain c. endospore stain d. simple stain

Gram stain. We also did a capsule stain and two wet mount

The Gram stain divides bacteria into two large groups:

Gram-positive and Gram-negative, based upon cell wall structure

What reagent is the mordant in the Gram Stain technique?

Iodine

what do you need to know and do for the midterm?

It will be 60 points there will be two parts: the practical part (14 pts) and the written part (46 pts) practical part: - one streak plate (4 points) and must incubate it the right way - one of the 4 differential stains (10 points). You will get a piece of paper saying which stain you have and will have to determine on your own which organism to use, one positive and the other negative for that particular stain. Then you will need to prepare your own smear, stain the smear, and view under a microscope. written part: - microscopy - cell morphology - stained cells (simple stained, gram stained, etc) - Structure (endospores, capsules, flagella) - Biofilm - Procedure related to stains, and application of the information - Will need to know names of bacteria mentioned in lab manual

You observed a stained slide under the microscope, and found some cells were random and rod shaped and the other cells were coccus shaped in chains. Was the smear made from a pure culture? Select one: a. Yes b. No

No

are the majority of organisms pathogenic?

No

In doing a Gram stain of the Gram standard you forget to rinse with 95% ethanol. What will you likely see? **all endospores are gram positive rods a. red cocci b. red spirals c. purple spirals d. purple cocci

The correct answer is: purple spirals, purple cocci

What must the condenser be set at to view your biofilm with phase contrast using the 40X objective.

PH2

What is an example of a biofilm

Plaque on the surface of your teeth

Because cells consist of mostly water, even under adequate magnification unstained cells may be difficult to see because of lack of contrast. What do we do to make them more visible?

Staining cells makes them more visible

Which of the following is the best explanation for using stains on smears. a. Staining allows you to better determine the colony morphology. b. Staining adds liquid to the cells to make them bigger and easier to see. c. Staining makes the smears smell better. d. Staining makes the cells visible by coloring them darker than the light background.

Staining makes the cells visible by coloring them darker than the light background.

Name gram positive organisms

Staphylococcus epidermidis **also Clostridium and Bacillus

Non acid fast bacteria

Spirosoma linguale, Escherichia coli, and Azotobacter vinelandii **all the gram neg ones

What is sporulation?

Sporulation is the process of endospore formation within a vegetative cell

From your streak plate, you will take an isolated colony to streak another plate. ________ of cells by transferring the colony to a new plate is commonly used by microbiologists to maintain cultures. This second plating will also allow any hidden cells of a different species to be diluted when streaking the plate for isolation. A colony of Staphylococcus epidermidis is very tiny and can easily hide within a larger colony of E. coli. In this course, you will deal with pure cultures each week. The same techniques performed for this experiment are used routinely in preparing these cultures. The reaction of each of these organisms to specific tests is known when they are prepared for you. If there is a positive result when the expected result is negative, this could mean the presence of _________. It is important to use proper aseptic technique when performing experiments so that you do not introduce contaminants from an unsterile loop, your hands, or the air, which would alter the results.

Subculturing contaminants

What is the appearance of colonies of Mycobacterium species? What is the reason for this?

The appearance is compact and wrinkled, due to the cell wall's hydrophobic nature.

You will make a smear for a Gram stain and inoculate a new plate from a colony on your streak plate. Select one: True False

The correct answer is 'True'.

Based on the results from the gram stain that you performed, which of the following description best describes the gram reaction of Spirosoma linguale and its appearance under the microscope? a. Gram positive; pinkish color, spiral shape b. Gram positive; purple color, spiral shape c. Gram negative; pinkish color, spiral shape d. Gram negative; purple color, spiral shape e. None of the above

The correct answer is: Gram negative; pinkish color, spiral shape

Yellow would describe the pigmentation of ______________________. Select one: a. Micrococcus luteus b. Bacillus cereus c. Escherichia coli d. Streptomyces coelicolor

The correct answer is: Micrococcus luteus (gram bacteria)

____________________ of cells by transferring the colony or a portion of a colony to a new plate is commonly used by microbiologists to maintain cultures. a. Gram staining b. Mixing c. Transformation d. Subculturing

The correct answer is: Subculturing

When does sporulation occur? a. when time is right to reproduce b. optimal growth conditions c. favorable environmental conditions d. harsh environmental conditions e. plenty of nutrients or water

The correct answer is: harsh environmental conditions

Mycobacterium's cell wall is best described as Select one: a. completely impermeable b. eukaryotic c. hydrophilic d. hydrophobic

The correct answer is: hydrophobic. The mycolic acids are hydrophobic

When a turf of flagella are attached at one end, the flagella arrangement is called _____________________. When a bacterium has one or more flagella at each end of the cell, the flagella arrangement is called __________________. a. lophotrichous; amphitrichous b. monotrichous; peritrichous c. peritrichous; lophotrichous d. amphitrichous; lophotrichous

The correct answer is: lophotrichous; amphitrichous

Which of the following is not needed for the endospore stain? Select one: a. malachite green b. methylene blue c. paper square d. microscope slide e. clothes pin

The correct answer is: methylene blue

Which of the following terms describe the whole colony shape of a colony? Select one: a. filamentous b. round c. umbonate d. lobate

The correct answer is: round (round, irregular, rhizoid)

what do differential stains do?

They utilize a combination of dyes to demonstrate a chemical or structural component of a cell.

A smear is the first step in making a simple stain. True False

True

When viewing prepared slides of stained flagellated bacteria, you must put oil on the coverslip to view with 100x. True False

True

Bacterial endospores of Bacillus stearothermophilus are useful in a laboratory setting because they are used to indicate the effectiveness of_______ . Bacillus stearothermophilus endospores are extremely heat resistant. Death of these spores indicates adequate killing ability by the autoclave. Because of the ability of these endospores to survive extreme adverse conditions, it is assumed that if they are killed, all other organisms will also be killed, rendering media or other items in the autoclave sterile

autoclaves.

give an example of pigmentation

beige white

The microscopes used in this class are_________ microscopes, which means that for each eye there is an ocular containing a magnifying lens. The total magnification of a microscope is the product of the magnification of the ocular lens and the magnification of the objective lens. The ocular lens on the lab microscopes is 10×. Therefore, the total magnification when using the 100× objective lens is 10× ∙ 100× = 1000×. This allows an invisible micron-sized cell to be visible through the microscope as a cell. Magnification is limited by a property of the lenses called resolution. Resolving power is the closest spacing between two points at which the points can still be seen as separate entities. Resolving power depends on the wavelength of light and a property of the objective lens called the numerical aperture. The maximum resolution in a compound light microscope is about 0.2 microns or micrometers (μm). Therefore, a cell must be at least 0.2 microns to be seen (though at this size, the cell would appear as a very tiny dot) and two cells must be at least 0.2 μm apart to be seen as separate cells.

binocular

Describe what the microscope slide will likely look like after being submerged for a week in the biofilm water sample.

cloudy and appear to have a film on it

what are the basic cell shapes we will see

cocci (spherical shaped) bacilli (rod shaped) spiral (spirillum)

unwanted organisms are called what?

contaminants

give two examples of elevation

convex umbonate

Endospores, in contrast, are said to be in a_________ state, in which no metabolic activities are occurring. If nutrients or water become depleted, or other environmental factors become detrimental to a vegetative cell, endospore formation will begin converting the vegetative cell into a single endospore. Endospores are ________ structures, not reproductive structures.

cryptobiotic survival

The primary stain used when performing a Gram stain is __________. Crystal violet enters the cells and stains them________. After excess crystal violet is rinsed from the smear with water, _______ is applied to the smear. Iodine acts as a________ binding to the crystal violet forming a complex not easily removed from Gram-positive cells. After rinsing away the iodine with water, ________ is added to the smear. Ethanol is a _________ capable of removing from cells basic dyes like crystal violet. Because the crystal violet is complexed with iodine, the ethanol is only capable of leaching out the crystal violet from __________ cells, leaving them colorless. After rinsing the ethanol off with water, a counterstain (a stain of another color) of __________ is added to the smear to stain the Gram-negative cells red.

crystal violet purple. iodine mordant ethanol decolorizer Gram-negative safranin

-To study microorganisms, it is helpful to be able to grow them. Microorganisms can be grown in the laboratory on or in a __________. The culture medium contains _________ such as carbon and nitrogen, required for the cells to grow. - what is a complex medium? -What is a defined medium?

culture medium nutrients -A complex medium is composed of digests of chemically undefined substances such as yeast and meat extracts or digests. A defined medium is one for which a precise chemical composition is known.

When using aseptic technique in performing a smear, what do you do with the inoculating loop after you have spread the culture on the slide? a. put the loop back in the culture b. rinse the loop with water c. dry heat sterilize the loop d. put the loop on the bench

dry heat sterilize the loop

give two examples of surface characteristics

dull shiny

If you see only one colony morphology on an agar plate, you can safely assume that only one type of organism is growing on the plate. True False

false

Immersion oil which is placed between the specimen and the objective increases magnification. True False

false

T or F: You may use both the coarse and fine focus adjustment knobs with the 100X objective. True False

false

A culture of Gram positive cells older than 72 hours produces the best Gram stains because older cells better retain the crystal violet bound to the iodine than younger cells.

false--gram positive stains over 24 hours old being to lose their primary stain and start to look gram negative

The Gram stain is a simple stain. Select one: True False

false. Its a differential stain

An endospore is a reproductive structure. Select one: True False

false. Its a survival structure

After you remove the cap from the tube, what is the correct order of events in demonstrating aseptic technique to prepare a smear from a broth culture? (You may use an answer choice more than once if needed. Your answer needs to be written with no spaces between letters; for example: abcdefgh) a. Replace the cap on the test tube b. Allow loop to cool c. Remove a loopful of microorganisms from the broth d. Heat the loop and wire until red hot e. Place a loopful of microorganisms onto the slide f. Flame the mouth of the culture tube g. Gently shake the broth culture to suspend the microorganisms

fcfaed

In prokaryotes, the flagellum is made of:

filament hook motor

Allergies have been bothering you lately. As you were inoculating your streak plate with E. coli you sneeze. With a plate in one hand and an inoculating loop in the other you were unable to quickly cover your mouth. You tried to sneeze into your arm but your unstifled sneeze was greater than your arm could shield. Following incubation of your plate, what do you expect to see on the plate next lab? a. Microorganisms are in the air transiently so there could be growth from contaminates in the air. b. There will be E. coli along with bacteria from your mouth on the plate. c. Nothing because the media in the plate was sterilized. d. Nothing because you did not inoculate the plate.

first 2. Microorganisms are in the air transiently so there could be growth from contaminates in the air., There will be E. coli along with bacteria from your mouth on the plate.

Preparing a smear is done using which of the following pieces of equipment? a. Microscope slide b. Bunsen burner or hot plate c. Inoculating loop d. sterile agar plate e. sterile swab f. computer

first 3. Microscope slide, Bunsen burner or hot plate, Inoculating loop

Heat ___________ kills the cells, destroying autolytic enzymes, and causes the cells to adhere to the slide.

fixation

remember, all endospores are what?

gram positive rods

Under what conditions does sporulation occur? Select one: a. optimal growth conditions b. plenty of nutrients or water c. favorable environmental conditions d. harsh environmental conditions e. when time is right to reproduce

harsh environmental conditions

Safranin a. stains Gram-negative bacteria in a Gram stain, vegetative cells in an endospore stain, and acid-fast cells in an acid-fast stain b. stains Gram-negative bacteria in a Gram stain and endospores in an endospre stain c. is a simple stain that stains most bacterial cells but not endospores or acid-fast cells d. is used as a counterstain in the Gram stain, endospore stain and acid-fast stain

is a simple stain that stains most bacterial cells but not endospores or acid-fast cells. It stains non endospore cells and gram neg

what are the types flagella (polar) can be:

monotrichous lophotrichous amphitrichous peritrichous

give two examples of optical properties

opaque translucent

Bacteria in biofilms communicate through:

quorum sensing

give two examples of whole colony shape

round irregular

Which of the following lists the counterstain, the mordant, the primary stain and the decolorizer of a Gram stain in this order? Select one: a. ethanol, crystal violet, iodine, safranin b. ethanol, iodine, crystal violet, safranin c. safranin, crystal violet, iodine, ethanol d. crystal violet, iodine, safranin, ethanol e. safranin, iodine, crystal violet, ethanol

safranin, iodine, crystal violet, ethanol

In the first stage of endospore formation, the replicated chromosome and a small amount of cytoplasm are isolated by a _______. The spore septum is simply an ingrowth of the plasma membrane. As endospore formation progresses, this will become a double-layered plasma membrane surrounding the genetic material and at this stage the structure is termed a__________. Peptidoglycan will be laid down between the two plasma membranes, and this layer is called the ________. Spore proteins form a ______ around the outside of the structure. The spore coat is responsible for the _______________. The peptidoglycan and spore coat each form thick layers. The ________ consists of the usual cell wall, cytoplasmic membrane, cytoplasm, and nucleoid. Endospores may be located in a_________ position within the vegetative cell or the sporangium may be swollen with a terminal endospore (Figure 4T-E).

spore septum. forespore cortex spore coat resistance of the endospore to harsh chemicals. core central, terminal, or subterminal

For this experiment, you will isolate pure cultures using solid media and a dilution technique called a ________. Using this technique, the bacterial cells in a broth culture will be diluted in order to separate the cells and isolate the organisms in pure culture as isolated colonies on the plate. From the number of different colony morphologies that appear on the plate, you can determine whether your broth culture contained more than one organism. If you only see one type of colony morphology on the plate, then your broth culture might have been a pure culture. Colony morphology is useful for identifying pure or mixed cultures, but many organisms have similar colony morphologies, so colony morphology alone is not sufficient to determine whether you have a pure or mixed culture. Another way of determining the purity of a broth culture is by _________. A simple stain can reveal the different cellular morphologies (shape and/or arrangement) of the microorganisms present, while a ___________like a Gram stain can reveal structural differences, helping you to determine the purity of a culture.

streak plate staining the sample. differential stain

What prevents a Gram negative bacteria from retaining the primary stain of a Gram stain? a. thick peptidoglycan layer b. thin peptidoglycan layer c. outer membrane d. cytoplasmic membrane e. none of the above

thin peptidoglycan layer

The first step in staining cells is what?

to make a smear

What is the purpose of inverting the agar plates while keeping in the incubator?

to prevent condensation pooling on streaked culture.

After performing a simple stain on your mixed broth culture, you will view with the microscope under 10x, then 100x with oil. True False

true

If we had grown E. coli on a plate with different media than used in class, we might have seen a change in colony size, shape, or color. Select one: True False

true

When you calculate population standard deviation, you take the square root of the summed squared differences divided by the total number of population observations. Select one: True False

true