Etiket Arşivleri: Pure Culture

Pure Culture Techniques ( Jackie Reynolds )

PURE CULTURE TECHNIQUES

Most specimens (from animal tissue, plant tissue, or environmental samples) will be mixed, with a variety of bacteria (or other microorganisms). A single gram of feces, for example, has over 1010 bacteria and that gram would have over 20 different bacterial species.
Particularly in a medical setting, where a patient’s body fluid or tissue (skin, blood, spinal fluid, urine) is sent to the microbiology lab for analysis, the specimens will most often be mixed. In order to identify the bacteria and run antibiotic susceptibilities on the causative agent of the infection, the microbiologist must be working with a pure culture of the bacterium. This exercise begins with a mixed culture of bacteria and will end with, hopefully, pure cultures of the 2 bacteria. Luckily, the 2 bacteria being used look different from each other when growing on agar plates. Two different types of agar plate isolation techniques will be used—streak and pour. Each method has advantages and disadvantages, and particular uses. The same mixed culture will be used for both methods. Another technique, called the
spread plate, is used commonly for counts, as is the pour plate technique. It uses pre-made agar plates, with the fluid inoculum being placed on top of the agar medium. The inoculum is then spread around on the plate with a bent glass rod. That procedure will not be done today. This also gives you a chance to see how agar plates are made. Some of the TSA plates are already made: others will be made by you. This requires that liquefied agar medium (sitting
in a water bath) be kept above the solidification temperature. Agar’s solidification temperature is 42 degrees C, but its liquefaction temperature is 100 degrees C. When sterilized in an autoclave or boiled, the medium will stay liquid until the temperature gets to 42 C. When that happens, the medium will solidify very fast. If it solidifies on you before you finish making your cultures, it has to be disposed of (if not used yet, it can be placed back in the sterile media racks).

REMEMBER

ALWAYS check agar plates carefully to make sure that there are no mold or bacterial contaminants on the plate: if so, discard the plate in the autoclave bag. If you see water running on the agar plate, you can do 2 things:
 Place the agar plate upside down in the 37C incubator with the top cracked.
 Get another agar plate.


Source: http://delrio.dcccd.edu/jreynolds/microbiology/2420/files/pure%20cultures.pdf

Isolation Technique

Isolation Technique

In nature microbial cultures are mixed

Identification relies upon isolating individual colonies

Testing requires pure cultures

As a result isolation technique provides an essential microbiological tool

Mixed Culture from Raw Poultry

Streak Plate Isolation Principle

An original inoculum containing a mixture of bacteria is spread into 4 quadrants on solid media.

The goal is to reduce the number of bacteria in each subsequent quadrant.

Colonies are masses of offspring from an individual cell therefore streaking attempts to separate individual cells.

Discrete colonies form as the individual cells are separated and then multiply to form isolated colonies in the later quadrants.

The Goal -Isolated Colonies to Start Pure Cultures

Can an isolated colony be considered pure?

This is generally assumed, however….

some colonies are very slow growers and may be too small to see.

some colonies may be growing under another colony

selective media may be preventing reproduction of some bacteria so they may be present but not visible

condensed water, capsules, slime, all represent areas where individual contaminant cells hide out.

Any special considerations?

Different species of microbes represent challenges….

Encapsulated bacteria are sticky and don’t separate well.

Some species are motile and do not stay where you streak them spreading across the plate.

Fungal spores easily contaminate cultures within a plate.

Organisms can gain entrance to a Petri dish through water or the edges, or from the air currents while you are streaking.

Microbes will surprise you each chance they get !

Isolation Requires Aseptic Technique

Isolation Requires Aseptic Technique

Streaking the Quadrants

Quadrant 1- Streak with broad narrow strokes in the upper half of the first quarter of the plate.

Incinerate and cool the loop between the quadrants

Quadrant 2 – Rotate the plate, enter the previous streak mark one or two times and then streak the upper portion of the second quarter of the plate with broad strokes.

Quadrant 3 – Rotate the plate, enter quadrant 2 one or two times and then streak with shorter more separated strokes from the top of the quadrant to the center.

Quadrant 4 – Enter quadrant 3 and then streak with broad S-shaped motions through the center of the plate.

Streaking the Quadrants

Isolated Colonies

Gram Stain

‎Laboratory‎ > The Techniques of Pure Cultures

Purpose

     Recognition of media which used  for cultivaring microorganisms and their preparation and sterilization. Learning of to colony selection techniques with pure culture techniques and inoculation of the cultures to different media by transferring techniques.

Theory

      The Techniques of Pure Cultures  

     Bacterial growth on/in a medium is called a culture. Microbiology laboratories work with pure cultures. A pure culture is when there is onlyone microorganism growing in/on the medium. The transferring of a bacterium from a stock culture to either a solid or liquid medium is called inoculation. Inoculating a bacterium on/in a sterile medium ensures the purity of a culture. Properly transferring a bacterium without contamination is called an aseptic transfer. Many steps are taken to ensure that neither the bacterium nor the medium is contaminated. Aseptic Techniques are the precautionary measures taken to prevent contamination of pure cultures and sterile laboratory equipment. Treat all organisms as potential pathogens. Many of the organisms can be opportunistic in their abilities to cause infection. Microorganisms in the lab atmosphere may come to rest on the desktop between classes and overnight, so disinfect lab top thoroughly before and after each lab period. This is accomplished by spraying the lab top down with a commercial disinfectant or a 10% bleach solution and allowing this to stand for a minute. You may then wipe down the bench with the paper towel.

Disinfection is the destruction or removal of infectious or harmful microorganisms from nonliving objects by physical or chemical methods. Heating process developed by Pasteur to disinfect beer and wines is called Pasteurization. It is still used to eliminate microorganisms from milk and beer. Not all microbes are destroyed by pasteurization. Chemicals used to disinfect objects are called disinfectants. When this treatment is directed at a living tissue, it is called antisepsis and the chemical is called antiseptic. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics.

Materials

·       Nutrient Agar

·       Nutrient broth

·       Autoclave

·       Beaker

·       Balon Tube

·       Distilled Water

·       Bunson Burner

·       Stirring magnet

·       Cotton

·       Rope

·       Flask

·       Test Tubes

·       Aliminium foil

·       Electrical heater

·       Sterile pipettes

·       Sterile petri dishes

·       Escherichia coli in Nutrient broth

·       Sterile Nutrient agar slant,plate and deep

·       Needle

·       İnoculation loop

·       Alcohol

·       Dilution tubes

Procedure

     Preparation of Nutrient broth: 3.2g of Nutrient broth media was taken and was placed in the beaker.A magnetic stirrer was added.After 400ml of distilled water was added in the beaker.İngredients was mixed.This mixture was distibuted into test tubes about 5-6ml for each tubes.Next, mouth of tubes were closed by

Results and Calculations

Discussion

İn this experiment we learned to prepare different media according to a certain microorganisms and a certain purposes. These media are anaerobic, synthetic, transport, enriched, selective, differential and microbiological assay media. For example; in anaerobic media; oxygen is removed from media with reducing agent, in synthetic media; their chemicals and concentration is known and identified, in transport media; microorganism is transferred from place to other place temporarily, in enriched media; number of scarce microorganisms are increased, but during this process growth of other microorganisms are not prevented, in selective media; growth of a special microorganisms is supplied and growth of other microorganisms are prevented, in differential media; appearance and size of microorganisms are determined with indicator, in microbiological assay media; concentration of some substances are measured. These media are prepared from nutrient broth and nutrient agar that broth represents liquid media and agar represents solid media.

Some media are slant media shape some media also are vertical shape. Slant media provide a large surface area so slant media is convenient for aerobic organisms, in vertical (deep) media, microorganism is cultivated toward the penetration of media and Petri plate also supplies a large surface area and because of this it supplies growth of microorganisms in a short time.

Finally; sterilization of media and equipment were learned during experiment. Moreover, required criteria one by one was told us. For sterilization, applied processes are classified as physical and chemical methods. Physical methods are heat, wet sterilization, tyndallization dry heat sterilization, radiation, freezing and bacteriological filtration. Chemical methods are salt, phenol and phenol derivatives, halogens, and alcohols. Chemical and physical methods and their effects on the microorganisms and equipment were learned and examined.

In this experiment, transfer of E. coli tried to learn with a certain transfer techniques, that these techniques are Broth to Broth, Broth to Slant, and Broth to Deep Transfer. Certain results were obtained from these transfers. For example; the formation of microorganisms were observed at broth to broth transfer and there was turbidity, at broth to slant transfer propagation of microorganisms were observed on the slant surface, at broth to deep transfer there were the formation of microorganisms but they were decreasing from above to deep, at slant to broth and at plate to broth transfers turbidity was observed and there were the formation of microorganisms. Meanwhile; petri plates were also examined against the formation of microorganisms, and at finger press; microorganisms were observed, another petri plate had been exposed to cough that a few microorganism was observed, and finally; at another petri plate finding a hair there was no the formation of microorganisms.

In here; we understood that nutrient agar slant and deep media are more suitable for preservation of microorganisms in a long time. In addition; when the microorganisms live they produce toxic materials and these toxic materials spread out fast in broth media. However; slant and deep media slow down the expansion of toxic materials. Because of this some microorganisms are preserved in the slant and deep media in order to protect a long time.

As a result, we say that we learned transfer techniques and, inoculum and incubation of microorganisms. In addition; we learned that when the transfers of microorganisms are transferred sterilization of media, environment and using apparatus must be taken care sufficiently, and must be rendered sterile.

Pure Culture

PURPOSE

The purpose of this experiment was to be demonstrated the techniques for transforming E. coli.

THEORY:

Bacterial growth on/in a medium is called a culture. Microbiology laboratories work with pure cultures. A pure culture is when there is onlyone microorganism growing in/on the medium. The transferring of a bacterium from a stock culture to either a solid or liquid medium is called inoculation. Inoculating a bacterium on/in a sterile medium ensures the purity of a culture. Properly transferring a bacterium without contamination is called an aseptic transfer. Many steps are taken to ensure that neither the bacterium nor the medium is contaminated. Aseptic Techniques are the precautionary measures taken to prevent contamination of pure cultures and sterile laboratory equipment. Treat all organisms as potential pathogens. Many of the organisms can be opportunistic in their abilities to cause infection. Microorganisms in the lab atmosphere may come to rest on the desktop between classes and overnight, so disinfect lab top thoroughly before and after each lab period. This is accomplished by spraying the lab top down with a commercial disinfectant or a 10% bleach solution and allowing this to stand for a minute. You may then wipe down the bench with the paper towel.

Disinfection is the destruction or removal of infectious or harmful microorganisms from nonliving objects by physical or chemical methods. Heating process developed by Pasteur to disinfect beer and wines is called Pasteurization. It is still used to eliminate microorganisms from milk and beer. Not all microbes are destroyed by pasteurization. Chemicals used to disinfect objects are calleddisinfectants. When this treatment is directed at a living tissue, it is called antisepsis and the chemical is called antiseptic. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics.

MATERIALS:

  • Nutrient Agar plate

  • Inoculating loop

  • Nutrient Agar slant

  • Inoculating needle

  • Nutrient Agar deep

  • Bunsen burner

  • Nutrient Broth

  • Test tubes and test tubes rack

  • Nutrient Broth of culture of E. coli

  • Etuv

  • Nutrient Agar plate of E. coli                        

  • lighter

PROCEDURE:

1- Microorganisms in environment;

a) Firstly; nutrient agar was taken, then the cover of a petri plate nutrient agar was removed and nutrient agar was exposed to air for5-10-20 minutes. Afterwards; cap of petri plate was replaced on the plate, and finally nutrient agar was incubated at 37 °C for 24 hours.

b) Again, another nutrient agar petri plate was taken petri plate was divided two parts underside by acetate pencil, and then we pressed slightly my finger on the nutrient agar medium, then it was incubated at 37 °C for 24 hours.

c) Again, another nutrient agar petri plate was taken and its cap was opened then one hair was put in the plate, after that; its cap was closed and it was incubated at 37 °C for 24 hours.

2- Transfer Techniques; 

i) Broth to Broth Transfer: Firstly; inoculating loop was taken and all of wire was heated to redness for sterilization. Afterwards, tube containing E. coli was taken my left hand and at the nozzle of tube, cotton was got out and the rim of tube was rendered sterile by flame. After that; inoculating loop was immersed to E. coli tube and E. coli was taken on the loop from nutrient broth. The rims of tube was held to re-flame, nozzle of tube was closed with cotton and preparing nutrient broth in a tube was taken and nozzle of the tube was rendered sterile with flame, and loop was immersed in the nutrient broth then nozzle of nutrient broth tube was held re-flame and was closed with cotton and nutrient broth was incubated at 37 °C for 24 hours.

ii) Broth to Slant Transfer: Firstly; inoculating loop was taken and all of wire was heated to redness for sterilization. Then, tube containing E. coli was taken my left hand and at the nozzle of tube, cotton was got out and the rim of tube was rendered sterile by flame. Next, inoculating loop was immersed to E. coli tube and E. coli was taken on the loop from nutrient broth containing E. coli. The nozzle of tube was held to re-flame, tube’s nozzle was closed by cotton again. Afterwards; our slant tube was taken and its cotton was removed, then nozzle of tube was held to flame in a nearly horizontal position. Next, loop containing E. coli was gone in a zigzag on the slant surface. E. coli was cultivated on the slant surface. Finally; nutrient slant tube was incubated at 37 °C for 24 hours.

iii) Broth to Deep Transfer: Firstly; inoculating needle was taken and all of wire was heated to redness for sterilization. Then, tube containing E. coli was taken my left hand and at the nozzle of tube, cotton was got out and the rim of tube was rendered sterile by flame. Next, inoculating needle was immersed to E. coli tube and E. coli was taken on the needle from nutrient broth containing E. coli. The nozzle of tube was held to re-flame, tube’s nozzle was closed by cotton again. Afterwards; our deep tube was taken and cotton was removed from tube and nozzle of tube was rendered sterile by flame. Needle containing E. coli was penetrated to deep nutrient agar tube, and then nutrient agar deep tube was incubated at 37 °C for 24 hours.

3- Agar Slant as source of inoculum; 

Slant to Broth Transfer: Firstly; inoculating loop was taken and was sterilized by flame as far as redness. Then tube containing E. coli was taken my left hand and cotton was got out at nozzle of tube. Next, the rim of tube was rendered sterile by flame. Then, inoculating loop was immersed to E. coli tube and E. coli was taken on the loop from nutrient slant containing E. coli and rim of tube was sterilized again, and cotton was closed the nozzle of tube again. Afterwards; our broth tube was taken, cotton was got out from nozzle of tube and nozzle of tube was rendered sterile by flame and loop containing E. coli was immersed to tube and was shaken, then nozzle of tube was held to re-flame and cotton was again closed , finally; nutrient broth tube was incubated at 37 °C for 24 hours.

4- Colony Selection;

In here; there was a nutrient agar petri plate containing E. coli and the cap of plate was opened carefully and E, coli was taken by sterilized loop in a short time. Afterwards; our nutrient broth was taken and cotton was got out, then nozzle of tube was rendered sterile by flame, and loop containing E. coli was immersed to nutrient broth, loop was shaken in the tube slightly. Next; nozzle of our nutrient broth tube was held to re-flame and was rendered sterile, cotton was closed. Finally; nutrient broth tube was incubated at 37 °C for 24 hours.

RESULTS:

Broth to Broth Transfer: There was turbidity and microorganisms were observed.

Broth to Slant Transfer: Propagation of microorganisms was observed on the surface.

Broth to Deep Transfer: There were microorganisms and they were observed toward top part more than toward bottom part.

Slant to Broth Transfer: There was turbidity, microorganisms were observed.

Plate to Broth Transfer: There was turbidity, microorganisms were observed.

Petri Plate (finger press): There were microorganisms, 11 small microorganisms were observed.

Petri Plate (air): 1 microorganism was observed at 5 minutes.

Petri Plate (one hair): no microorganisms.

DISCUSSION:

In this experiment, transfer of E. coli tried to learn with a certain transfer techniques, that these techniques are Broth to Broth, Broth to Slant, and Broth to Deep Transfer. Certain results were obtained from these transfers. For example; the formation of microorganisms were observed at broth to broth transfer and there was turbidity, at broth to slant transfer propagation of microorganisms were observed on the slant surface, at broth to deep transfer there were the formation of microorganisms but they were decreasing from above to deep, at slant to broth and at plate to broth transfers turbidity was observed and there were the formation of microorganisms. Meanwhile; petri plates were also examined against the formation of microorganisms, and at finger press; twelve small microorganisms were observed, another petri plate had been exposed to air for 5 minutes that one microorganism was observed, and finally; at another petri plate finding a hair there was no the formation of microorganisms.

In here; we understood that nutrient agar slant and deep media are more suitable for preservation of microorganisms in a long time. In addition; when the microorganisms live they produce toxic materials and these toxic materials spread out fast in broth media. However; slant and deep media slow down the expansion of toxic materials. Because of this some microorganisms are preserved in the slant and deep media in order to protect a long time.

As a result, we say that we learned transfer techniques and, inoculum and incubation of microorganisms. In addition; we learned that when the transfers of microorganisms are transferred sterilization of media, environment and using apparatus must be taken care sufficiently, and must be rendered sterile.