Question

please answer all the questions for microbiology :
1. Be able to draw and/or identify general bacterial cell shapes and cell organizations

2. Know how to do a streak for isolation, and a spread plate and why you would do each

3. how to do a Gram stain, Endospore stain and acid fast stain and why you would do each.

4. Know the tools of the microbiologist and what they are used for

5. Be able to explain the results of the hand-wash experiment

6. Be able to identify and describe the results of the antiseptics/disinfectants, antibiotics experiment and interpret the results

7. Know what aseptic technique is and how to exhibit aseptic technique

Answer 1

1. On the basis of their morphology and shape bacteria can be categorized as: Unicellular Bacteria,Spirochaetes, Myxobacteria and Filamentous Bacteria (Actinomycetes).

Unicellular bacteria are further divided into-

• Cocci (or coccus for a single cell) are round cells, sometimes slightly flattened when they are adjacent to one another.
• Bacilli (or bacillus for a single cell) are rod-shaped bacteria.
• Spirilla (or spirillum for a single cell) are curved bacteria which can range from a gently curved shape to a corkscrew-like spiral. Many spirilla are rigid and capable of movement. A special group of spirilla known as spirochetes are long, slender, and flexible.
• 

2.

Procedure of Spread Plate Technique

1. prepare a dilution series from a sample.
2. Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate.
3. Dip the L-shaped glass spreader into the alcohol.
4. Flame the glass spreader (hockey stick) over a Bunsen burner.
5. Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating the Petridish underneath at the same time.
6. Incubate the plate at 37°C for 24 hours.
7. Calculate the CFU value of the sample. Once you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/mL in the original sample.

Uses of Spread Plate Technique

1. It is in use for viable plate counts, in which the total number of colony forming units on a single plate is enumerated.
2. It is used in order to calculate the concentration of cells in the tube from which the sample was plated.
3. It is routinely used in enrichment, selection, and screening experiments.

Procedure

1. Sterilize the inoculating loop in the bunsen burner by putting the loop into the flame until it is red hot. Allow it to cool.
2. Pick isolated colony from the agar plate culture and spread it over the first quadrant (approximately 1/4 of the plate) using close parallel streaks or Insert your loop into the tube/culture bottle and remove some inoculum.
3. Immediately streak the inoculating loop very gently over a quarter of the plate using a back and forth motion (see area 1 in the figure below).
4. Flame the loop again and allow it to cool. Going back to the edge of area 1 that you just streaked, extend the streaks into the second quarter of the plate (area 2).
5. Flame the loop again and allow it to cool down . Going back to the area that you just streaked (area 2), extend the streaks into the third quarter of the plate (area 3).
6. Flame the loop again and allow it to cool. Going back to the area that you just streaked (area 3), extend the streaks into the center fourth of the plate (area 4).
7. Flame your loop once again.

Importance of streaking

• To help produce isolated colonies of an organism (mostly bacteria) on an agar plate. This is useful when we need to separate organisms in a mixed culture (to purify/isolate particular strain from contaminants) or when we need to study the colony morphology of an organism.
• Identify the organism: biochemical tests to identify bacteria are only valid when performed on pure cultures.

3

Gram Stain Procedure

1. Place slide with heat fixed smear on staining tray.
   
2. Gently flood smear with crystal violet and let stand for 1 minute.
   
3. Than tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
   
4. Gently flood the smear with Gram’s iodine and let stand for 1 minute.
   
5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The smear will appear as a purple circle on the slide.
   
6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize.
   
7. Immediately rinse with water.
   
8. Gently flood with safranin to counter-stain and let stand for 45 seconds.
   
9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
   
10. Blot dry the slide with bibulous paper.
    
11. View the smear using a light-microscope under oil-immersion.

.

Procedure of endospore stain:

1. Prepare smears of organisms to be tested for presence of endospores on a clean microscope slide and air dry it.
2. Heat fix the smear.
3. Place a small piece of blotting paper (absorbent paper) over the smear and place the slide (smear side up) on a  wire gauze on a ring stand.
4. Heat the slide gently till it starts to evaporate (either by putting the slide on a staining rack that has been placed over a boiling water bath or via bunsen burner).
5. Remove the heat and reheat the slide as needed to keep the slide steaming for about 3-5 minutes. As the paper begins to dry add a drop or two of malachite green to keep it moist, but don’t add so much at one time that the temperature is appreciably reduced.
   No overheating The process is steaming and not baking.
6. After 5 minutes remove the slide from the rack using a clothespin
7. Remove the blotting paper and allow the slide to cool to room temperature for 2 minutes.
8. Rinse the slide thoroughly with tap water (to  wash the malachite green from both sides of the microscope slide).
9. Stain the smear with safranin for 2 minutes.
10. Rinse both side of the slide to remove the secondary stain and blot the slide/ air dry.

Procedure of Acid-Fast Stain

1. Prepare the bacterial smear on clean and grease free slide, using sterile technique.
2. Allow smear to air dry and then heat fix.
   Alcohol-xation: recommended when the smear has not been prepared from sodium hypochlorite (bleach) treated sputum and will not be stained immediately. M. tuberculosis is killed by bleach and during the staining process. Heat-xation of untreated sputum will not kill M. tuberculosis whereas alcohol-xation is bactericidal.
3. Cover the smear with carbol fuchsin stain.
4. Heat the stain until vapour just begins to rise (i.e. about 60 C). Do not overheat. Allow the heated stain to remain on the slide for 5 minutes.
   Heating the stain: Great care must be taken when heating the carbol fuchsin especially if staining is carried out over a tray or other container in which highly ammable chemicals have collected from previous staining. Only a small ame should be applied under the slides using an ignited swab previously dampened with a few drops of acid alcohol or 70% v/v ethanol or methanol. Do not use a large ethanol soaked swab because this is a re risk.
5. Wash off the stain with clean water.
   Note: When the tap water is not clean, wash the smear with ltered water or clean boiled rainwater.
6. Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear is sufciently decolorized, i.e. pale pink.
   Caution: Acid alcohol is ammable, therefore use it with care well away from an open ame.
7. Wash well with clean water.
8. Cover the smear with malachite green stain for 1–2 minutes, using the longer time when the smear is thin.
9. Wash off the stain with clean water.
10. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not blot dry).
11. Examine the smear microscopically, using the 100 X oil immersion objective.

Petri Dishes

In order to run their tests, scientists need samples of microorganisms, and they need containers in which to hold and study them. Cultures of bacteria are placed in petri dishes or on plates. These containers are made of clear acrylic, circular in shape, with a lip around the edge to prevent a culture from spreading outside the dish. Petri plates also have clear acrylic lids as some bacteria produce airborne spores that travel through our atmosphere and, if breathed in, can be harmful.

Agar

Agar is a food or gel substance added to petri dishes to provide nutrients that bacteria need to grow. Agar can be one of several different materials used for this purpose, including blood, chocolate, and tryptic soy.

Other Culture Tools

To move culture materials, scientists use one or more other tools. One of these tools is called a swab or loop. The loop is a thin metal rod, several inches long with a small metal loop attached at the end. The loop is swiped across the culture to collect microbes to be transferred to another agar-filled petri dish. Pipettes are also used, but instead of removing microbes with a scrape, they have light suction power. The top of the pipette is squeezed while the bottom is placed in the culture. When the top is let go, suction power sucks up microbes into the pipette's hollow shaft.

Microscope

Powerful laboratory microscopes allow microbiologists to examine their cultures at close range since the human eye is obviously limited when it comes to seeing bacteria growing or moving. There are specialized microscopes that help scientists study even the smallest of viruses, such as electron and fluorescent microscopes.

5.

The results demonstrate that handwashing with non-antibacterial soap is much more effective in removing bacteria from hands than handwashing with water only. Although handwashing with water alone reduced the presence of bacteria on hands substantially, the study supports the policy of many current hand hygiene campaigns promoting the use of soap . The strong association between hand hygiene method and bacterial contamination of hands found in our study suggests that the prevalence of faecal indicator bacteria may also be used to monitor changes in hygiene behaviour in the general population, for example following hygiene promotion campaigns.

Hygiene behaviour is difficult to measure because people tend to change their behaviour under observation or over-report desired practices . Results were positive for bacteria of potential faecal origin were more common in people frequently shaking hands, reporting soil contact or those scoring low on a hygiene score based on self-report [9]. The microbiological method used in this and our earlier studies is relatively simple

6. Results of the antiseptics/disinfectants

It is clear that microorganisms can adapt to a variety of environmental physical and chemical conditions, and it is therefore not surprising that resistance to extensively used antiseptics and disinfectants has been reported. Of the mechanisms that have been studied, the most significant are clearly intrinsic, in particular the ability to sporulate, adaptation of pseudomonads, and the protective effects of biofilms. In these cases, “resistance” may be incorrectly used and “tolerance,” defined as developmental or protective effects that permit microorganisms to survive in the presence of an active agent, may be more correct. Many of these reports of resistance have often paralleled issues including inadequate cleaning, incorrect product use, or ineffective infection control practices, which cannot be underestimated. Some acquired mechanisms (in particular with heavy-metal resistance) have also been shown to be clinically significant, but in most cases the results have been speculative. Increased MICs have been confirmed, in particular for staphylococci. However, few reports have further investigated increased bactericidal concentrations or actual use dilutions of products, which in many cases contain significantly higher concentrations of biocides, or formulation attributes, which can increase product efficacy; in a number of cases, changes in the MICs have actually been shown not to be significant . Efflux mechanisms are known to be important in antibiotic resistance, but it is questionable if the observed increased MICs of biocides could have clinical implications for hard-surface or topical disinfection. It has been speculated that low-level resistance may aid in the survival of microorganisms at residual levels of antiseptics and disinfectants; any possible clinical significance of this remains to be tested. With growing concerns about the development of biocide resistance and cross-resistance with antibiotics, it is clear that clinical isolates should be under continual surveillance and possible mechanisms should be investigated.

It is also clear that antiseptic and disinfectant products can vary significantly, despite containing similar levels of biocides, which underlines the need for close inspection of efficacy claims and adequate test methodology. In addition, a particular antiseptic or disinfectant product may be better selected (as part of infection control practices) based on particular circumstances or nosocomial outbreaks; for example, certain active agents are clearly more efficacious against gram-positive than gram-negative bacteria, and C. difficile (despite the intrinsic resistance of spores) may be effectively controlled physically by adequate cleaning with QAC-based products.

In conclusion, a great deal remains to be learned about the mode of action of antiseptics and disinfectants. Although significant progress has been made with bacterial investigations, a greater understanding of these mechanisms is clearly lacking for other infectious agents. Studies of the mechanisms of action of and microbial resistance to antiseptics and disinfectants are thus not merely of academic significance. They are associated with the more efficient use of these agents clinically and with the potential design of newer, more effective compounds and products.

Results of antibiotics

Like the older criteria that were in use up to the present, the new EUCAST/DIN criteria for the assessment of antibiotic effectiveness in the tripartite classification scheme—"susceptible," "intermediate," and "resistant"—are independent of the particular indication for which the antibiotic is to be used. For a number of drugs, however, the indications for which the drug is approved will have an influence on the breakpoints. Thus, an antibiotic that has been approved solely for the treatment of urinary tract infections should be assessed differently from one that is also used to treat respiratory tract infections. When the breakpoints were set for Neisseria meningitidis, for example, account was taken of the fact that this organism usually causes an infection in the central nervous system. The new standards (ISO, EUCAST/DIN) introduce a new level of complexity to the determination of antibiotic resistance of infectious microorganisms. The newly standardized breakpoints across Europe will, however, improve the reliability of categorization as "susceptible," "intermediate," and "resistant." This in no way relieves treating physicians of the duty to consider the available therapeutic options carefully when prescribing antibiotics.

7.

Sterile work area

The simplest and most economical way to reduce contamination from airborne particles and aerosols (e.g., dust, spores, shed skin, sneezing) is to use a cell culture hood.

• The cell culture hood should be properly set up and be located in an area that is restricted to cell culture that is free from drafts from doors, windows, and other equipment, and with no through traffic.
• The work surface should be uncluttered and contain only items required for a particular procedure; it should not be used as a storage area.
• Before and after use, the work surface should be disinfected thoroughly, and the surrounding areas and equipment should be cleaned routinely.
• For routine cleaning, wipe the work surface with 70% ethanol before and during work, especially after any spillage.
• You may use ultraviolet light to sterilize the air and exposed work surfaces in the cell culture hood between uses.
• Using a Bunsen burner for flaming is not necessary nor is it recommended in a cell culture hood.
• Leave the cell culture hood running at all times, turning it off only when they will not be used for extended periods of time.

Good personal hygiene

Wash your hands before and after working with cell cultures.

In addition to protecting you from hazardous materials, wearing personal protective equipment also reduces the probability of contamination from shed skin as well as dirt and dust from your clothes.

Sterile reagents & media

Commercial reagents and media undergo strict quality control to ensure their sterility, but they can become contaminated while handling. Follow the guidelines below for sterile handling to avoid contaminating them. Always sterilize any reagents, media, or solutions prepared in the laboratory using the appropriate sterilization procedure (e.g., autoclave, sterile filter).

Sterile handling

• Always wipe your hands and your work area with 70% ethanol.
• Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them in the cell culture hood.
• Avoid pouring media and reagents directly from bottles or flasks.
• Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids, and use each pipette only once to avoid cross contamination. Do not unwrap sterile pipettes until they are to be used. Keep your pipettes at your work area.
• Always cap the bottles and flasks after use and seal multi-well plates with tape or place them in resealable bags to prevent microorganisms and airborn contaminants from gaining entry.
• Never uncover a sterile flask, bottle, petri dish, etc. until the instant you are ready to use it and never leave it open to the environment. Return the cover as soon as you are finished.
• If you remove a cap or cover, and have to put it down on the work surface, place the cap with opening facing down.
• Use only sterile glassware and other equipment.
• Be careful not to talk, sing, or whistle when you are performing sterile procedures.
• Perform your experiments as rapidly as possible to minimize contamination.