Preparation of Media and Reagents & Aseptic Technique and Pure Culture Essay Sample

Preparation of Media and Reagents & Aseptic Technique and Pure Culture Pages
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Methods in molecular biology involve the use of living organisms (generally bacteria) and a wide variety of reagents, enzymes, and DNA molecules. The bacterial used in recombinant DNA techniques must be cultivated on media that allows them to grow optimally. Most bacteriological media contain rich sources of nutrients such as tryptones or peptones (which are rich sources of amino acids), yeast extract (which is a rich source of vitamins and other nutrients), and various salts. Because many of the bacterial strains used in molecules biology harbor plasmids that confer resistance to one or more antibiotics, antibiotics are commonly added to media. Similarly, other additives are often added to media so that various phenotypic changes in the organisms can be visualized by color reactions. for example, when a gene is cloned into an appropriate vector (for example pUC18), bacteria that harbor a fragment of foreign DNA can be distinguished from those that do not by adding a chromogenic substrate (X-gal) to the media.

Most bacteriological media are prepared in either a liquid form (broth) or in a solid form (agars, Fig. 1). Meticulous care is taken to provide the proper concentration of nutrients, pH, and others factors needed for the cultivation of microorganisms. Agar is a product of a marine alga that is a no growth substrate for most bacteria, does not melt until it reaches 100 C, and one molten, does not solidify until about 44 C. Agar plates allow the formation of isolated colonies of bacteria, while liquid media allow the growth of large amounts of cells for procedures such as DNA isolations. Since the ingredients and glassware employed in the preparation of media can contain unwanted microorganisms, freshly prepared media must be sterilized (e.g. autoclaving or filtration) immediately. An autoclave is essentially a large pressure cooker that heats the media to a temperature of 121 C by injecting steam under pressure into the autoclave chamber. The pressure prevents the media from boiling at the elevated temperature. Filtration is carried out by using 0.2 m membrane filters. This is necessary as some media ingredients (such as antibiotics) will break down under autoclave temperature and they are added to media from filter-sterilized stocks after autoclaving.

Molecular biology procedures also require a wide variety of chemical reagents. These contain a myriad of components, but common features of all molecules biology reagents are that they are generally autoclaved to destroy any DNAses present

Aseptic technique is required to transfer pure cultures and to maintain the sterility of media and solutions. By aseptic technique the biotechnologist takes prudent precautions to prevent contamination of the culture or solutions by unwanted microbes. Many of the Petri dishes and tissue culture plates used for growing pure cultures or microorganisms are made of plastic and come presterilized from the manufacturer, filling these vessels with a sterile medium requires the used of aseptic technique. Proper aseptic technique also protects the biotechnologist from contamination with the culture, which should always be treated as a potential pathogen. Aseptic technique involves avoiding any contact of the pure culture, sterile medium, and sterile surfaces of the growth vessel with contaminating microorganisms.

Typical steps for transferring a culture from one vessel to another are:

1. flaming the transfer loop

2. opening and flaming the mouth of the cultures tubes

3. picking up some of the culture growth and transferring it to the fresh medium

4. flaming the mouths of the culture vessels and resealing them

5. reframing the inoculating loop

The introduction of microorganisms into the culture medium is called inoculation.

The utilization of microbes in biotechnology depends on pure cultures, which consists of only a single species, and the maintenance of the purity of the isolates through subsequent manipulations. Three techniques will be performed: the streak plate, the pour plate, and the spread plate. This technique will allow single colonies to be separated on the agar.

Materials

LB broth (liquid)

LB agar

Nichrome loop

Petri dishes

E. coli culture

Fire flame

Method

Preparation of media and reagents

Produce A – LB broth and LB agar

The ingredients are weighed out according to the volume specified by the instructor (as in Table 1). The appropriate volume of distilled water are measured and poured it into a shake flask. The weighed amount of ingredients slowly added to the beaker with slow stirring. The pH is adjusted to 7.4 (0.1) with 4 N NaOH. The pH checked with a pH meter or pH paper. For making LB agar: the required amount of agar is weighed [at a concentration of 1.5% (w/v)] and added it to the LB broth. The flask of medium covered with aluminium foil and autoclave for 15 minutes at 121C.

Produce B – Pouring LB agar plates (LB agar or Lb agar/AP)

The plates are labelled on the bottom (along the edge with a fine-point permanent marker) to indicate the medium prepared. The flask is removed from the water bath. The outside is wiped carefully with a paper towel. The neck of the flask of agar is flamed and the required amount of medium are poured into the plate. Each plate should contain 20 – 25 ml of medium. The plates are allows to set. The surface of the plates must be dry by overnight incubation at room temperature or by opening the plates and placing them, medium surface down in an oven at 45 – 55C. The lids should also face downwards separately from the base of the plate. The plates are leaved at 45 – 55C for 15 min. The agar plates can be stored for at least a few weeks at 4C following wrapping in parafilm or sealing in a plastic bag.

Table: Medium for culturing E. coli strain

Medium

Volume required

Amount to be weighed

Usage

LB broth

1000 ml

10.0 g tryptone

5.0 g yeast extract

10.0 g NaCl

For bacterial growth

LB agar

1000 ml

10.0 g tryptone

5.0 g yeast extract

10.0 g NaCl

5.0 g Bacto agar

Preparation of agar plates/slants

Aseptic technique and pure culture

Produce A – Quadrant streak plate technique

5 ml of LB broth with E. coli was inoculated and grew at 37 C. The bottom of the sterile Petri dishes was labeled with name, date, streaking method used and temperature incubation. The Nichrome loop was flamed until it was red-hot. The loop is allowed to cool. Some of E. coli was removed and transfer to the agar on the Petri dishes by different streaking method. The plate was incubate and inverted at 37 C. The plate was then examined for colony morphology and presence of possible contamination.

Streaking method

Produce B – Pour plate technique

The bottom of sterile Petri dishes was labeled name, date and temperature of incubation. Melt agar was used as a medium. The Nichrome loop was flamed till red hot and let to be cool down. A loopful of the culture is transfer to the melt agar medium. The content in the melt agar was stirred and pour into the Petri dishes. The agar was let to be hardened. The pour plate was incubating in an inverted position. The plate was examine and the appearance was describe

Produce C – dilution series and the spread plate technique

Six tubes containing 9 ml of sterile dilution blanks were prepared. The tubes are labeled 1, 2, 3, 4, 5, 6 and 7. 1 ml pf the test culture were took and added to tube 1. This was the 10-1 dilution. The tube was vortex to mix the cells. 1 ml from tube 1 was taken out by fresh pipette and added to tube2. This was the 10-2 dilution. These operations were repeated from tube 2 to tube 3 and so on down the series until tube 7. The Nichrome loop was flamed until red hot and let to be cool down. A loopful of the culture is transfer to a agar surface and was spread evenly over the plate. This method was repeated for remaining tube. After that those plate was incubate overnight at 37 C in a invest position. After one night, the colonies from was count and then colony forming unit (CFU)/ml was calculated using following formula:

Colony forming (CFUs)/ml (original sample) = (average number of colonies/plate) X dilution factor.

Calculations

Molarity = mol/L

Weight % (w/w) = (weight solute/weight solution) X 100%

Volume % (v/v) = (volume solute/volume solution) X 100%

Weight/volume % (w/v) = [weight solute (g)/volume solution (ml)] X 100%

Normality (N) = no meq A/no mL solution

= no eq A/no L solution

Q1

LB agar = 1.5% (w/v) agar

1.5 g in 100 ml

X g in 1000 ml

15g in 1000 ml

(15/1000) X 100% = 1.5%

Q2

Ampicillin add into agar to a final concentration = 50g/ml

LB agar = 1L = 1000ml

50 g in 1 ml

X g in 1000 ml

50000 g in 1000 ml

(50000g/1000ml) X 100% = 50g/ml

Q3

LB medium = 1000ml

Tryptone = 10g = X% (w/v)

Tryptone = 10g in 1000ml medium

(10/1000ml) X 100% = 1% (w/v)

Yeast extract = 5g = X% (w/v)

Yeast extract = 5g in 1000ml medium

(5g/1000ml) X 100% = 0.5% (w/v)

NaCl = X% (w/v)

NaCl = 10g in 1000ml medium

(10g/1000ml) X 100% = 1% (w/v)

Ampicillin = X (20mg/ml)

ampicillin = 20mg in 1 ml (final concentration)

X g in 1000ml

(20mg/1ml) X 1000ml = 20g

Result

Broth liquid media

The liquid media such as broth become cloudy because the bacteria are present. This could be the result of only one bacterial cell entering the medium, then dividing repeatedly to produce million. Thus the broth turns from clear to cloudy in color.

Pour plate

In this case, we mix the E. coli with the liquid medium before we pour the liquid medium in to the Petri dish to let it become solid. Therefore, we can observe that the bacterial was growth within the medium and not at the surface of the medium.

Streak plate

In streak plate, the bacteria are streak over the large surface area of the plate, thus the amount is diluted to the individual cell at the end point of streaking, as this bacterial cell multiply repeatedly the single colony will observed

Streak plate

Colony forming (CFUs)/ml (original sample) = (average number of colonies/plate) X dilution factor.

Colony forming (CFUs) = (average number of colonies/plate) X (dilution factor) X (original sample, ml)

= (183) X 106 X 10000 l

= 1.83 X 1012 colonies per original sample

Question

Preparation of media and reagents

1. How does nutrient agar different from nutrient broth?

Nutrient agar is the jelly to which food has been added for the growth of microbes, the solid version of nutrient both supplemented with agar. Nutrient agar is also a mixture of agar and beef extract and proteins that allow many microorganisms to grow in culture in other case. The nutrient broth is a general purpose liquid basal medium composed of beef extract and peptone which allows many types of microorganism to grow.

2. Why is important to prevent any condensation falling on the agar surface?

The agar plates are inverted during incubation and the purpose of placing the plates upside down is to prevent the condensation from dripping down onto the agar surface which can then facilitate the movement of organisms between colonies that might collect on the lids from falling back on agar and causing colonies to run together. If this occurs, the single colonies are hardly to be observed.

3. How do the process of wiping the flask containing the sterile agar and flaming the mouth of the flask of broth or agar prevent contamination?

The both wiping and flaming processes can prevent contamination. In the wiping process, the flask containing the sterile agar is wipe by the sterile water, this is needed because wiping can remove the residual that stick on the flask thus remove the unwanted microorganism and result the sterile condition and prevent contamination. In the flaming process, the mouth of the flask is flame over the Bunsen burner, when near the flame, the heat will generated and this can kill and destroy the unwanted microorganism thus sterile condition occur and no contamination. They may also sterilize instruments using an alcohol. Aseptic technique also protects the student microbiologist from contamination with the microbe, which should be always treated as a potential pathogen.

4. Explain the usage of both defined and undefined media for the growth of bacteria and yeast.

Defined medium being synthesized from the individual chemicals as required by the organism so that the exact molecular composition is known, while the undefined medium or complex media is made up of natural product such as yeast extract, contains at least one component that is neither purified nor completely characterized nor even completely consistent from batch to batch, often these are partially digested proteins from various organism sources. For example, nutrient broth is derived from culture of yeast.

Aseptic technique and pure culture

1. What is the purpose of inverting inoculated plates during incubation?

The inoculated plates are inverted during incubation is to prevent falling of other microorganisms on the medium surface due to gravity and also to prevent the condensation dripping onto the agar surface which can facilitate the movement of organism between the colonies.

2. Can you explain how both the streak and pour plate methods effected separation of the bacteria?

For streak plate method, the bacteria grow along the line draw with inoculating loop. The bacteria are not randomly dispersed. While for pour plate method, bacteria are randomly dispersed. In streak plate, the bacteria are streak over the large surface area of the plate, thus the amount is diluted to the individual cell at the end point of streaking, as this bacterial cell multiply repeatedly the single colony will observed. While in pour plate, the bacterial is distributed evenly in the liquid form of agar, so it amount is diluted, as a result it will grow as individual colonies

3. Where should colonies appear in the case of streak plates and pour plates?

For streak plate, the colonies was observed appear on the surface, while for pour plate, the colonies was appear within the agar plate.

4. What factor (s) could account for an absence of growth on a pour plate and streak plate?

In pour plate and streak plate, the absence of growth can be caused by inoculating when the loop was still in high temperature, this will kill of all the E. coli. Besides that, the small part of the culture that removed might not contain any bacteria as the bacteria might not randomly disperse in the culture media.

5. What explanation could be given for the failure of obtaining isolated colonies on a streak plate?

In streak plate, isolated colonies are not found sometimes. This is due to the amount of bacteria that transfer into media while inoculating is in enormous amount. This will cause the striking of the colonies, where only turbid lines will been observed, but single colonies are hardly to be observed.

6. List two errors associated with serial dilutions.

During dilutions, errors might occur and this will affect the amount of bacteria in the media. As bacteria might not being evenly distributed in the test tubes, the dilutions carried out might not be the dilution factors intended. Also the use of micropipettor with tips from different manufacturer will also influence the dilutions. In addition, instrumental error of micropipettor also can result in 0.1 l deviations from intended, thus affecting the dilution process.

7. Why are different pipettes used between dilutions?

Different pipettes used between dilutions are to prevent contamination with excess bacteria with each time diluting the media.

8. Observe the plates that you spread with the diluted E. coli culture. If you prepared the dilutions correctly, you should have roughly tenfold colony on each plate as you go to the higher dilutions. Is this case with your plates? If not, suggests an explanation.

The colonies of bacteria in the cultures media do not reduce by 10 times with each 10 times dilution. This is because some error was occurred during the serial dilution. For instance, bacteria are not evenly dispersed during dilution. Beside that, different pressure applied on micropipettors during sucking of media containing bacteria also can result in different amount of bacteria introduced into the nest dilutions test tube.

9. How can you be certain that you have a pure culture on an agar plate?

The certain that we have a pure culture on an agar plate, the appearance of the surface of medium are observed. In this case, E. coli cause will cause turbidity on its colonies with milky white color. Different appearance often denotes the presence of other microorganism. Therefore, we expect a pure culture to have a standard, same color (milky white) and sounded shape, randomly dispersed colonies of E. coli.

Conclusion

There are 1.83 X 1012 colonies per original sample and there are also many pre caution step that we have to take care during the medium/reagent preparation and also for the aseptic technique.

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