Micro lab

Halophiles

"salt-loving" archaea that live in environments that have very high salt concentrations

2. Why is it tricky to stain flagella?

Bacterial flagella are normally too thin to be seen under such conditions. The flagella stains employs a mordant to coat the flagella with stain until they are thick enough to be seen

Glucose positive

Bc it turned yellow

SERIAL DILUTION-SPREAD PLATE ISOLATION TECHNIQUE

Ethanol flame rod spreader

Anaerobes

Microorganisms that can grow in the absence of oxygen are called anaerobes.

Type of microbes likely to see in the fridge?

Psychrophiles refers to microbes that can grow best at relatively low temperatures, between 0oC and 20oC. In general, few bacterial species are psychrophilic, and most fungi grow better at cooler temperatures, this is why we see fungi growing in our refrigerated foods more often than bacteria. There are also many psychrophilic algae that grow on glaciers or snow fields and it is these algae that give snow a reddish tint that some refer to as "watermelon snow".

What dye is used to detect oxygen by appearing red and lack of oxygen by being colorless?

Resazurin

Lab 4, how environment effects growth

The growth rate of a microbial population is affected by many factors, including structural, physiological and environmental factors. Environmental factors that influence the growth rate of microbes include: oxygen, temperature, osmotic pressure, pH, UV radiation and CO2 concentration. In this week's lab we will observe the effect of temperature, osmotic pressure, pH and UV irradiation on the bacterial growth.

Many fungi and some bacteria grow optimally in acidic environments, they are called?.

acidophiles

Gram stain procedure

1. Prepare heat-fixed bacterial smears. 2. apply 1 or 2 drops of crystal violet dye to each of the smears on the slide. 3. Wait 60 seconds 4. Rinse with water indirectly 5. Apply 1 or 2 drops of Gram's iodine (the mordant) wait 60 seconds 6. Water 7. Decolorizing- 1 or 2 drops of alcohol to each of the smears on the slide to decolorize the gram-negative cells. 10 seconds 8. Water 9. 1 or 2 drops of safranin (the counterstain) to each of the smears on the slide. Wait 60 seconds 10. Water. wipe off the back (non-stained side) of the slide with a Kimwipe or lab tissue wipe. 11. Dry the stained slide by placing on the slide warmer, stained side up, for 1 min or let it air dry a few minutes.

How many times more acidic is lemon juice (pH 2) compared to tomatoes (pH 4)?

100 times The pH scale is the negative base 10 logarithm of the hydrogen ion concentration. Thus, a pH of 2 means [H+]= 10-2 while a pH of 4 means [H+]= 10-4. Thus, lemon juice is 100 (102) times more acidic than tomato juice.

Areobes

Aerobes are microorganisms that use oxygen for their respiration.

Which is more productive? Aerobes of anaerobic?

Aerobic metabolism is usually more productive because it produces more ATP. Aerobic cultures usually grow faster than anaerobic ones.

Lactose negative

Bc no color change (our unk is red) itd turn yellow

Differential Stains

Differential Stains: The Gram-Stain Differential stains allow microbiologists to distinguish between different groups of bacteria based on their reaction with the stain. The two most common differential stains are the Gram stain and the Acid-Fast stain. Both of these differentiate between bacteria based on the composition of the cell wall.

Acid fast stains can be used to detect?

Leprosy and Tuberculosis

What is mutagenic (causes DNA mutations) but at high levels or for extended periods of time it can be microbicidal (kill microbes).?

Ultraviolet radiation?

acid fast stain

a differential stain used to identify bacteria that are not decolorized by acid-alcohol

Escherichia Coli

gram negative rod gram-negative, so they stain pink

Most microbes prefer to grow at pH near neutral, they are

neutralophiles

Smear technique

- Spread a thin film made from a liquid suspension of cells on a slide - Allow the slide to air dry - Heat fixing • Heat the slide gently after it has been air dried Put drops of water on slide. ONE colony from the agar plate and transfer the bacteria onto the water on the slide. Spread the bacteria around the entire slide. 6. Allow the smear on the slide to briefly air dry on the bench, don't wait more than 2 or 3 minutes for this. If they are not totally dry after 2-3 minutes, just go on to the next step. 7. Heat-fix the cells onto the slide by setting the slide with the bacteria side facing up on the slide warmer for 5 minutes. Heat fixing causes the cells to stick to the slide (so that they do not get washed off during staining), kills the cells and makes them more permeable to the dye. But be careful because overheating them causes them to distort and lyse. 8. Allow the slide to cool before going on to the staining procedure.

Flagella Stain

1 Slide with pre-prepared smear of Proteus sp. ​​​​1 Ampule of Flagella stain ​​​​Large glass petri dish for rinsing 1. Place the slide on the staining rack. 2. Holding the ampule of Flagella stain with the dropper tip pointing down over your slide, gently squeeze the middle to break open the glass ampule inside. DO NOT REMOVE THE DROPPER TIP FROM THE AMPULE!!! 4. Squeeze the ampule to apply stain onto the slide, filling the wax circle (3-6 drops). Discard the used ampule in a Biohazardous Sharps container. 5. Let sit 4 minutes. A film will form over the stain. 6. Fill a large glass petri dish with water and gently put the slide into it. Gently rinse by running the water in the dish, but not directly on the slide. The film will wash off. Remove the slide from the water and let it air dry. DO NOT BLOT, WIPE OR HEAT THE STAINED SMEAR! Remember these smears are NOT heat fixed and can easily come off the slide if touched. 7. Observe the slide under the microscope. View first with the 4X objective lens to position the slide. Move the slide so the outer part of the stained smear is in the center of your field of view. See Figure 3.5. Once you have positioned the slide appropriately, move to higher magnification lenses. Flagella will appear as long filaments attached to the cells and will be stained violet.

capsule stain procedure

1. Begin w/ drop of serum & add Congo red stain 2. Add organisms & emulsify 3. Use 2nd clean slide to draw drop across to other end 4. Air-dry (do NOT heat-fix) 5. Flood w/ Maneval's stain for 1 min. then rinse 6. Blot dry w/ Bibulous paper

Gram stain

A staining method that distinguishes between two different kinds of bacterial cell walls, gram positive and gram negative The two most important differences are that gram-negative cells have a much thinner peptidoglycan layer and an outer membrane composed of phospholipids. These two differences are responsible for their different reactions during the gram-staining procedure.

Agar

Agar is a polymer made up of galactose (sugar) subunits. It is derived from agarose, which is a polysaccharide and forms a supporting structure in the cell walls of certain algae. It is a gel like substance obtained from genera Gracilaria and Gellidium. Linear polysaccharide agarose and agaropectin actually make up agar. A growth medium is provided by the agar plate, which consists of agar along with other nutrients. Agar is indigestible by most of the bacteria. Agar is a mixture of two components: the linear polysaccharide agarose, and a heterogeneous mixture of smaller molecules called agaropectin.[2] It forms the supporting structure in the cell walls of certain species of algae, and is released on boiling. These algae are known as agarophytes, and belong to the Rhodophyta (red algae) phylum. Most Microbes don't eat it High melting point.

fermentation (anaerobic respiration)

An important alternative to respiration is the primary biochemical pathway of many anaerobes, which produces ATP from glucose without utilizing O2 through a biochemical pathway known as fermentation. Therefore, fermentation can be described as anaerobic catabolism of glucose. However, a lot fewer ATP's are formed per glucose molecule via fermentation compared to respiration. Only two ATP's per glucose are obtained from fermentations, compared to up to 38 from aerobic respiration. Therefore a lot more cellular energy can be produced from respiration than fermentation. As in respiration, the fermentative pathway also begins with glycolysis. However, during fermentation, the pyruvate that is produced from glycolysis does not enter the TCA cycle. Instead, it is directly converted into one or more of the possible fermentative products; acids, gases or alcohols. The specific acid, gas or alcohol that is produced depends on the microbe. Figure 5.4 shows the possible fermentation products that various microbial species can produce.

endospore stain

Bacterial endospores are highly resistant to hostile physical and chemical conditions. These are a dormant form of the bacterium that allows it to survive sub-optimal environmental conditions. Because these spores are resistant to heat, radiation, disinfectants, and desiccation, they are difficult to eliminate from medical and pharmaceutical materials and are a frequent cause of contamination. Only a few genera of bacteria such as Bacillus and Clostridium are capable of forming endospores. Figure 3.4. Endospore stain. The vegetative cells stain red and the spores stain green. Spores have a tough outer covering made of keratin and are highly resistant to heat and chemicals. The keratin also resists staining, so specialized procedures are necessary to stain endospores: 1. Malachite green stain is forced into the spore by heating the cells. 2. Vegetative cells are then decolorized with water and stained pink with spore safranin counterstain. Endospores may be located in the middle of the cells (central), at the end (terminal), or between the end and the middle of the cells (subterminal). The endospores themselves may be round or oval. Some spores may also be free of cells and just appear as green ovals. Red stained rods, green stained speers

Capsule

Capsule stain of Streptococcus pyogenes. Some bacteria produce a structured extracellular matrix, generally composed of polysaccharide (there are exceptions), referred to as a capsule. The presence of a capsule is often associated with the ability to cause disease, with capsule producing strains often being more virulent than non-encapsulated strains. Griffith's experiment with Streptococcus pneumoniae is a prime example of enhanced virulence of a pathogen that is encapsulated over those that are non-encapsulated. Capsules may act as adhesins to attach to host tissue, but most frequently confer an ability to evade the immune response. The capsule cannot be stained by methods involving heat as the capsule is highly hydrated and will shrink dramatically when heated or subjected to other procedures involved in the staining process. To overcome these problems a simple negative staining procedure is available. Negative stains stain the background, but not the cells. The unstained cells can be visualized as a silhouette against a dark background. You will not be doing capsule staining yourselves today, but be sure to observe the demonstration provided and document your observations. The background should appear grey-black; the capsule colorless, and the cells gray to red in color. Note the size of the capsule relative to the size of the bacterium.

How are cells able to keep oxidative by-products from turning macro molecules off? What are the two enzymes? What do they turn the by-products into?

Cells that are able to live in the presence of oxygen have evolved enzymes to cope with H2O2 and O2- and thus are not inactivated. Superoxide dismutase (SOD) is an enzyme that catalyzes the destruction of the by-product O2-. This enzyme is so critical to aerobic life that oxygen-metabolizing cells possess multiple SOD isoforms to protect them against harmful effects of the superoxide radical. Catalases are heme-containing proteins that catalyze the conversion of hydrogen peroxide (H2O2) to water and molecular oxygen. These two antioxidant enzymes together protect cells from the destructive effects of ROS. The reactions that they each catalyze are summarized in figure 5.2 below. 2O2- + 2H. ➡️ O2 + H2O2 Superoxide Dismutase 2H2O2​​→​2H2O + O2 Catalase Figure 5.2 The neutralization of ROS by superoxide dismutase and catalase

Chocolate Agar

Complex medium used to culture fastidious bacteria, particularly those found in clinical specimens. Not selective or differential.

1. Why is it necessary to heat the slide continuously during the endospore staining process?

Cooking the malachite green into the endospore Wall bc they are resistance to the hostile environment because of keratin.

Effect of Temperature: Observations: In the table below record the relative amount of growth of each organism at the various temperatures. Use three plus signs (+++) for the heaviest growth, two plus signs (++) for moderate growth, one plus sign for light growth and a zero (0) for no growth. Make conclusions about the temperature optimum of the microbes. Choose from the following terms: Psychrophile, Mesophile or Thermophile and Thermotolerant

E Coli: mesophile Growth conditions: Temperature range: 4- 45°C (39-113°F); can survive refrigeration and freezing. Optimum Temperature: 37°C (98.6°F) P. Fluorescence is a Psychrophile B. Stearothermophilus is a thermophile

3. What protects most bacteria from swelling up and bursting in hypotonic solutions?

Exoskeleton(cell wall) which provides the bacteria with several benefits. This cell wall protects the bacterium from damage by encircling it with a tough, rigid structure. The primary function of the cell wall is to maintain the cell shape and prevent bursting from osmotic pressure, so this bursting process only occurs in animal and protozoa cells which do not have cell walls. Read more on Brainly.com - https://brainly.com/question/5505953#readmore

What's an alternate to respiration that would be without the use of oxygen to produce ATP?

Fermentation

Flagella

Flagella Add substance to thicken flagella to be visible. Some bacteria are motile by means of flagella (flagellum is singular form). This motility allows the bacterium the ability to find nutrients or move away from toxic compounds. The positioning of the flagella (monotrichous, amphitrichous, peritrichous, or lophotrichous) (Figure 3.3) is a useful taxonomic characteristic. The difficulty in visualizing flagella with the ordinary light microscope is that they are below the resolving power of the microscope. Flagella consist of a single protein filament with a diameter of about 15 nm. To overcome this problem, a mordant is precipitated onto the filament, increasing its diameter; the coated filament is then stained. It is important to use young cultures when doing this stain. Slide to stain was preprepared bc it's fragile.

Lab 4 procedure OBJECTIVES: To learn how various environmental factors affect the growth of microbes.

Get 3 TSA plates and divide each plate into three labeled sections to place: Pseudomonas fluorescens broth ​​​​​Escherichia coli broth ​​​​​Bacillus stearothermophilus broth label one plate 4oC, another 37oC and the third one 55oC and incubate them at those respected temperatures

S. epidermidis Staphylococcus epidermidis

Gram positive

Broth Agar deep Agar slant Agar plate

In general, broth tubes are used when we want large numbers of cells for use in a single application. A test tube with nutrient agar is called an agar deep and it is most useful for growing microbes with low or zero oxygen requirements (anaerobes) deeply within the agar. An agar slant is a test tube of nutrient agar that was allowed to solidify at an angle creating a larger flat surface for growth at the top of the agar. Agar slants are most useful for growing maintenance cultures of microbes that we want to have on hand for extended periods. An agar plate is a Petri plate containing nutrient agar that provides the largest surface area for growth of microorganisms.

Citric Acid Cycle (Krebs Cycle)

In oxygen respiring organisms, glycolysis is followed by the citric acid cycle, which produces the electron carriers, NADH and FADH2. In the final stage of respiration, the electrons of NADH and FADH2 are fed into the electron transport chain. In the final reaction in the electron transport chain, the electrons are transferred to a final electron acceptor. In aerobic respiration, molecular oxygen (O2) is the final electron acceptor but some microbes can respire anaerobically by using alternative final electron acceptors such as Nitrate (NO3-), ferric iron (Fe3+), sulfate (SO42-), or carbonate (CO32-). Less energy is released with these alternate acceptors than with O2 because they don't have as high a reduction potential (Eo') as O2. Respiration, whether aerobic or anaerobic, is coupled to the production of ATP by an enzyme complex called ATP synthase. The final ATP yield from the complete aerobic oxidation of a single molecule of glucose is 38 ATP molecules. However, the yield is lower if an alternative electron acceptor is used anaerobically.

streak plate method (isolation technique)

In the streak plate method an inoculation loop is used to spread the cells from the mixed culture over the surface of an agar plate. By streaking the loop containing the bacterial cells many times over the entire surface of the agar plate without crossing the same section of the plate more than once, the cells are continuously diluted such that eventually some sections of the plate are inoculated with a small enough inoculum to produce widely separated colonies (See Figure 1.6). Cells streaked in this way grow on the surface of the agar and are easily accessible for transferring onto fresh nutrient media to establish the pure culture.

Effects of UV irradiation T-soy Agar plate ​​​​​1 - Sterile cotton swab ​​​​​Escherichia coli broth Share the culture with the other students on your table. 1. Label the TSA plates with your names, E.C. for E. coli and "UV". Divide the plates in half and label one half of one plate 0 sec, the other half of that plate 30 sec, label one half of the other plate 60 sec and the other half of that second plate 120 sec. 2. Soak the sterile cotton swab with E. coli from the broth culture and swab the entire surface area of both plates. You want to inoculate the entire plate to get a "lawn" of bacteria. You can use the same cotton swab for both plates and you can dip the swab into the E. coli broth more than once if needed. Dispose of the swab in the biohazardous waste container. 3. Bring the plates to the fume hood and put on some protective goggles. 4. Place one plate under the UV lamp and remove the lid, YOU MUST NOT FORGET TO REMOVE THE LID. 5. Place or hold an index card over half of plate and then turn on the UV lamp. Start counting the time and irradiate each side of the plate for the appropriate length of time as indicated by the labels on your plates. Repeat this with the other plate. 6. Put the lids back on the plates and place the plates in the appropriate area in the back of the laboratory. 7. Next week we will evaluate bacterial death due to UV radiation by comparing the results of the different irradiation times.

It killed more the longer under uv light.

What are the microbes that cause most human diseases?

Mesophiles Microbes that grow best at intermediate temperatures, between 20oC and 40oC, are called mesophiles. Since body temperature of humans is 37oC, most of the microbes that can cause human disease are mesophiles.

Three Circle technique

Place three circles of water onto the slide using the glass dropper bottle. They should each be made with a couple of drops of water, so that they don't dry out before you get a chance to place the microbes on them. 4. Hold the inoculation loop in your dominant hand like you would hold a pencil. Flame the inoculation loop by holding it in the flame until the wire turns red. Wait for it to cool slightly. 5. Using the sterile inoculation loop obtain ONE coloy from the agar and transfer the bacteria onto spot #1 on the slide (the circle closest to the frosted edge) by spreading the loopful of bacteria onto the drop of water while making a circle about the size of a dime. This smear should be thick and cloudy. 6. Flame the loop and wait for it to cool slightly. 7. Obtain a small amount of organisms from the first spot on the slide and transfer them onto spot #2 by spreading the loopful of bacteria onto the drop of water while making a circle about the size of a dime. This smear should be a bit cloudy but not too thick. 8. Flame the loop again and wait for it to cool slightly. 9. Obtain a small amount of organisms from the second spot on the slide and transfer them onto spot #3 by spreading the loopful of bacteria onto the drop of water while making a circle about the size of a dime. This smear should be visible but not too thick or clumpy. 10. Allow the smears on the slide to briefly air dry on the bench, don't wait more than 2 or 3 minutes for this. If they are not totally dry after 2-3 minutes, just go on to the next step. 11. Heat-fix the cells onto the slide by setting the slide with the bacteria side facing up on the slide warmer for 5 minutes. Heat fixing causes the cells to stick to the slide (so that they do not get washed off during staining), kills the cells and makes them more permeable to the dye. But be careful because overheating them causes them to distort and lyse. 12. Allow the slide to cool before going on to the staining procedure

What does Phile refer to? What does tolerant refer to? What does duric refer to?

Please note that the suffix "phile" refers to organisms that grow optimally at the specific conditions referred by the prefix, whereas the suffices "tolerant" or "duric" refer to organisms that can survive those specific conditions but do to grow well or do not grow at all in them. For example, most bacteria are psychrotolerant, they survive cold environments but they do not thrive in them. Only a few species are psychophilic, that thrive and grow optimally in cold environments. The same can be said for the other environmental conditions that we are dealing with this week. A halophile grows optimally in high salt concentrations but a halotolerant organism simply survives those conditions but does not grow in them very well.

Acid fast stain procedure

Procedure 2: Acid-fast Stain PER PAIR OF STUDENTS: 1 culture of Mycobacterium sp. ​​​​ 1 culture of Staphylococcus epidermidis ​​​​ Carbolfuchsin KF stain ​​​​ Acid Ethanol ​​​​ Methylene Blue stain 1. Prepare and heat-fix a mixed smear containing both of the bacterial cultures together. Label the slide on the frosted edge. Using a wax pencil, mark a circular region about the size of a quarter on the middle of the slide. 2. With a sterile inoculating loop touch a very small amount of S. epidermidis in the middle of the circled area. Sterilize the loop, let it cool, pick a colony of Mycobacterium and transfer it to the middle of the slide. BE VERY CAREFUL TRANSFERING THE MYCOBACTERIUM! 3. Add one small drop of water in the circled area (do NOT touch the dropper to the surface). Carefully use a toothpick to break up the colony as much as possible and then let the slide air-dry. 4. Heat-fix the slide for 10 mins on the slide warmer and let it cool. 5. Add 2-3 drops of Carbolfuchsin KF stain to the circled area and let it sit for 5 mins. 6. Rinse the slide under gently running water, 5-10 seconds. 7. Leave the water running slightly, add 2-3 drops of acid alcohol to decolorize and IMMEDIATELY rinse well with water. 8. Counterstain with methylene blue for 30 seconds. 9. Rinse with water. Shake off excess water into the sink and wipe off the back of the slide (non-stained side) with a Kimwipe. Remove excess water by placing on the slide warmer for 1 min or let air dry completely. 10. Observe stain. Acid-fast bacteria will appear red, while non acid-fast bacteria appear blue/purple.

acid fast stain procedure

Procedure 2: Acid-fast Stain PER PAIR OF STUDENTS: 1 culture of Mycobacterium sp. ​​​​ 1 culture of Staphylococcus epidermidis ​​​​ Carbolfuchsin KF stain ​​​​ Acid Ethanol ​​​​ Methylene Blue stain 1. Prepare and heat-fix a mixed smear containing both of the bacterial cultures together. mark a circular region about the size of a quarter on the middle of the slide. 2. With a sterile inoculating loop touch a very small amount of S. epidermidis in the middle of the circled area. Sterilize the loop, let it cool, pick a colony of Mycobacterium and transfer it to the middle of the slide. BE VERY CAREFUL TRANSFERING THE MYCOBACTERIUM! 3. Add one small drop of water in the circled area (do NOT touch the dropper to the surface). Carefully use a toothpick to break up the colony as much as possible and then let the slide air-dry. 4. Heat-fix the slide for 10 mins on the slide warmer and let it cool. 5. Add 2-3 drops of Carbolfuchsin KF stain to the circled area and let it sit for 5 mins. 6. Rinse the slide under gently running water, 5-10 seconds. 7. Leave the water running slightly, add 2-3 drops of acid alcohol to decolorize and IMMEDIATELY rinse well with water. 8. Counterstain with methylene blue for 30 seconds. 9. Rinse with water. Shake off excess water into the sink and wipe off the back of the slide (non-stained side) with a Kimwipe. Remove excess water by placing on the slide warmer for 1 min or let air dry completely. 10. Observe stain. Acid-fast bacteria will appear red, while non acid-fast bacteria appear blue/purple.

Endospore stain procedure

Procedure 3: Endospore Stain PER STUDENT PAIR:​1 culture of Bacillus sp. ​​​1 culture of Corynebacterium xerosis ​​​Endospore Stain Supplies It is important to use cultures that are over 24 hours old to really be able to observe good endospore production. 1. Make a smear of each bacterium and heat fix the slide as done previously for Gram staining. 2. Place the slide in a glass petri dish with the smear side facing up. Put petri dish on slide warmer. 3. Cover the smear with a small piece of paper towel, NOT hanging over the edges of the slide. 4. Cover the smear and paper towel with several drops of malachite green stain, and steam. Don't let the slide get dry--keep adding a couple drops of stain if it appears to approach dryness. Steam gently for about 5-10 minutes. REASON: You are cooking the malachite green into the endospore wall, as they are very resistant to staining. 5. Remove the petri dish from heat and dispose of the green paper in the wastebasket using forceps. Wash the slide gently in running water about 20 seconds, getting all the extra green stain off the slide. 6. Counterstain with spore safranin stain for one minute. Gently rinse with water, shake off excess water into the sink and wipe off the back (non-stained side) of the slide with a Kimwipe. REASON: The running water washed the green stain out of the vegetative cells, and they became colorless. The counterstain now dyes them red. 7. Remove excess water by placing on the slide warmer for 1 min or let it air dry completely. Examine with oil immersion optics. Observe red vegetative cells, and green endospores and free spores. Observations: Make accurate drawings of your observations for each stain that you performed/observed today. Include as much detail as possible including color.

Agar

Solid media has all the required nutrients, and it is usually identical in composition to broth but a solidifying agent, called agar, is added to the mixture. Solid media is therefore referred to as nutrient agar. The agar itself does not supply any nutrients to the medium and acts only to solidify it. Agar is obtained from the cell wall of some species of marine algae and it is sometimes also used to solidify foods such as yogurt, ice cream, jams and jellies. Agar is an ideal solidifying agent for microbial growth media because it is indigestible which means that the microbes do not utilize it as a nutrient source they only utilize the specific nutrients that are in the media. Also, agar remains solid at the temperatures that are often used to grow microbes, 37oC and 25oC. A special property of agar is that its melting temperature is very different from its solidifying temperature. Agar melts at 100oC but does not solidify until it is cooled to about 42oC. This property of agar allows us to use a microbial isolation technique called the pour-plate technique in which the microbes are put into the agar while it is still semi-liquid but not so hot that it kills them.

Why do we use thioglycollate agar to determine oxygen requirements of the microbe?

Thioglycollate binds up free oxygen creating a reducing environment in the medium. A dye, resazurin, is used as an indicator of the presence of oxygen. Resazurin is red in the presence of oxygen and colorless under anaerobic conditions. After inoculation and incubation, oxygen will diffuse into the top part of the medium and support aerobic growth. Immediately below the pink resazurin zone, there is a low level of oxygen. Growth in that zone suggests the presence of a microaerophile. Growth all the way at the bottom is indicative of anaerobic growth.

Simple stain

The Simple Stain: In the simple staining technique a basic dye such as methylene blue, crystal violet or safranin is used to stain the cell's cytoplasm. These dyes dissociate in solution to form positively charged molecules plus a negatively charged ion. Since the cellular cytoplasm has a net negative charge the positively charged dye molecule is attracted to the negative charges in the cytoplasm. The cells are washed with water and the dye is retained within the cytoplasm by the charge interactions. Acidic dyes, such as congo red, that have a negative charge are repelled by the negatively-charged cytoplasm, so acidic dyes are often used to stain the cells' background rather than the cells themselves. This is called a negative stain.

Gram stain procedure

The gram-stain technique consists of four steps. In the first step the cells are exposed to a primary dye, crystal violet, which is basic and is therefore attracted to the cell cytoplasm by virtue of its charge. Crystal violet stains all the cells a deep purple color. In the second step a mordant, Gram's iodine, is added. The mordant is a stabilizer that causes the primary dye to form large crystals in the peptidoglycan meshwork of the cell wall. The thicker peptidoglycan layer of gram-positive cells entraps the violet crystals much more effectively than the thinner peptidoglycan layer of gram-negative cells. After Gram's iodine, all of the cells still have a deep purple color. Alcohol added in the third step dissolves the outer membrane of gram-negative cells causing them to lose the violet crystals but has no effect on the gram-positive cells. After the third step the gram-positive cells remain purple while the gram-negative cells are colorless. The second and third steps are therefore the key differentiating steps in the gram-staining procedure because these are the steps in which the reaction depends on the biochemical composition of the cell wall. In the fourth and final step, the colorless gram-negative cells are counterstained with a secondary dye, safranin, in order to make them visible once again. Safranin is also a basic dye which stains the cells pink, but safranin can only stain cells that are not already stained. Therefore, at the end of the gram-staining procedure, gram-positive cells look purple and gram-negative cells look pink. The gram-stain does not always produce reliable results. An important factor affecting the reliability of the gram-stain is the age of the cultures. 24-48 hour culture best results for staining

acid fast stain LAB 3: STAINING OF ACID-FAST BACTERIA AND STAINING OF BACTERIAL CAPSULES, ENDOSPORES, AND FLAGELLA

The majority of bacteria are stainable by the Gram stain. However, some genera such as Mycobacterium and Nocardia are resistant to the dye and can only be visualized by the acid-fast stain. Although most Mycobacterium sp. are saprophytes, two species M. tuberculosis and M. leprae are important human pathogens. The acid-fast stain is an important tool in the diagnosis of diseases caused by these bacteria.

Acid/neutral/base review

The pH (partial hydrogen concentration) of a solution is determined by the concentration of H+ ions and is defined as the negative log to the base 10 of the H+ ion concentration. Solutions with a high concentration of H+ ions have low concentration of OH- ions, these solutions have low pH and are said to be acidic. On the other hand, solutions with low [H+] and high [OH-] have high pH and are said to be alkaline or basic. Certain solutes have the ability change the pH of aqueous solutions. Solutes that decrease the pH of a solution are called acids and those that increase the pH of a solution are called bases. The pH scale goes from 0 to 14, thus solutions with a pH exactly at the mid-point, pH = 7, have equal [H+] and [OH-] and are said to have neutral pH. Pure water has exactly the same concentrations of H+ and OH- ions and thus the pH of pure water is 7, perfectly neutral. Because the pH scale represents logarithmic numbers, each change of one pH value is 10 times more or less acidic. For example, a solution of pH 6 is 10 times more acidic than a solution with pH 7 and 100 times more acidic than a solution of pH 8. Figure 4.3 shows the pH of common substances.

3. How might a bacterium benefit from having a capsule?

This layer is disease-causing. This layer is well organized and not easily washed off. It is usually composed of polysaccharides, but could be composed of other materials (e.g., polypeptide in B. anthracis). Because the capsule helps to protect bacteria against phagocytosis, the capsule is considered a virulence factor. A capsule-specific antibody may be required for phagocytosis to occur. Capsules also contain water which protects bacteria against desiccation. They also exclude bacterial viruses and most hydrophobic toxic materials such as detergents. The capsule is found most commonly among gram-negative bacteria, such as E.coli. However, some gram-positive bacteria may also have a capsule. B.megaterium for example, synthesizes a capsule composed of polypeptide and polysaccharides and S. pyogenes synthesizes a hyaluronic acid capsule. Vaccination using capsular material is effective against some organisms (e.g., H. influenzae type b and S. pneumoniae).

Others are ________, able to survive but not multiply in acid.

acidoduric (or acidotolerant)

What by-products are we looking for in the lab to determine if it has the ability to ferment?

acids, gases

Microbes that prefer to grow at high pH are referred to as.

alkalophiles

Aerotolerant anaerobes

always perform anaerobic metabolism but they are not killed in the presence of oxygen.

faculative anaerobes

are capable of both aerobic and anaerobic metabolism. They metabolize as aerobes if oxygen is present and as anaerobes when oxygen is not present.

obligate anaerobes

are killed in the presence of oxygen and can only grow in its absence.

microaerophiles

are microbes that grow best in low concentrations of oxygen.

aseptic technique

aseptic technique. Aseptic technique is any procedure that 1) protects your cultures from being contaminated with unwanted organisms and 2) protects you and the environment around you from being contaminated with the microbes that you are working with.

Why are the oxidative by-products a problem?

because they react readily with all cellular macromolecules and inactivate them.

acid fast stain

carbolfuchsin used to stain Mycobacterium species A staining procedure for identifying bacteria that have a waxy cell wall. (Have lipids, mycolic acids on surface of acid fast bacteria making it waxy) These problems can be overcome by applying moderate heat or detergents to soften lipid components of the cell wall and allow the stain to penetrate. Once stained, theses cells resist decolorization with acid-alcohol (3% HCl in 95% ethanol); hence the name "acid-fast." Mycolic acids are a group of complex branched-chain hydroxyl lipids. The carboxylic acid group must be free (unesterified) to react on a 1-to-1 basis with fuchsin dye. Mycolic acids are complexed to peptidoglycan of mycobacterial cell wall and this complex somehow prevents the passage of the acid-alcohol solvent during decolorization.

How is ultraviolet radiation useful in microbiology?

helpful for sterilization and disinfection purposes, but its use is limited because it does not penetrate deeply into proteinaceous matter (such as blood or sputum) and it does not pass through glass or plastic. Only surfaces and air are effectively decontaminated with UV radiation. A few microorganisms grow optimally in the presence of UV light but most do not.

The Kinyoun (cold acid-fast)

method is used for demonstrating acid-fast properties of the genus Mycobacterium. Like the Gram stain, the acid-fast stain is a multistep process involving three different reagents. 1) Carbolfuchsin stain is allowed to penetrate into the cell. Phenol, which is present in the stain, enhances penetration of the stain into lipids. 2) Decolorizing agent is acid-alcohol (3% HCl + 95% Ethanol) 3) Counterstain is methylene blue. Stained cells that have lost the primary stain will stain blue.

Phenol red

pH indicator, turns yellow under acidic conditions.

Types of stains

simple, differential, structural

Gram stain procedure

smear, heat fix, stain with crystal violet 1 min, wash, apply grams iodine, wash, apply 95%alcohol drop by drop, wash, counterstain safranin, sec. wash, blot dry, examine.

What is ROS?

superoxide (O2-) and hydrogen peroxide (H2O2), collectively known as reactive oxygen species

What are the by-products of oxygen in aerobic activity?

superoxide (O2-) and hydrogen peroxide (H2O2), collectively known as reactive oxygen species (ROS).

Glycolysis

the first biochemical pathway in the oxidation of glucose, is composed of 10 chemical reactions which convert glucose to pyruvate. The reactions of glycolysis do not directly require oxygen, so they are carried out by aerobic and anaerobic organisms. In oxygen respiring organisms, glycolysis is followed by the citric acid cycle

Bacterial smears

two procedures for preparing bacterial smears on microscopes slides. You can choose between using the smear technique or the three-circle technique for preparing the bacterial smears.

What does the Saccharomyces cerevisiae feement? What causes the bubbles in beer?

yeast Saccharomyces cerevisiae ferments sugar and the pyruvate is converted into alcohol and carbon dioxide. The carbon dioxide causes the bubbles in the beer. Acids produced by fermentative bacteria also give the characteristic taste to may food products including milk, sour cream, buttermilk and yogurt and the acids also cause the solid components of the milk to coagulate giving these products their creamy texture. Bacteria present in milk ferment lactose which is used in the production of all cheeses. Blue and Roquefort cheeses are flavored by allowing a blue-green mold in the genus Penicillium to grow on the cheese. Many other products including wine, liqueurs, soy sauce and olives are also produced by fermentative bacteria or fungi. Microbial fermentation is therefore an economically extremely important industry.