Some tips to remember:. A portion of a 10 mL graduated pipette is shown in Figure 6. What is the volume of liquid in this pipette? Solid and semi-solid media. Growing cultures of bacteria on solid media agar plate or slant permits us to view and identify colonial characteristics, and also provides a way to separate bacteria in a mixed culture. Bacteria may be grown in agar slant or stab media in tubes if the purpose is to maintain them in a longer term culture. Generally, bacteria grown on slants will remain viable for a few weeks to a few months, and sometimes longer if stored in a refrigerator.
In this laboratory, you will be introduced to aseptic techniques and basic lab skills needed to grow and maintain bacteria in culture. You will be applying these skills often, so mastery is important. Below, write the names of the two bacteria in the mixed culture and the appropriate BSL, as specified by your instructor:.
The techniques needed will first be demonstrated by your instructor. Using aseptic technique, use a 10 ml graduated pipette to transfer 2 ml of broth to each tube. As demonstrated, use a flame-sterilized inoculating loop to pick up from the surface of the M. Note how the broths look immediately after you inoculate them they should still look mostly clear. Bacterial growth in broths is indicated by the development of a cloudy appearance. If the newly inoculated broth looks cloudy at the start, you will have no way to determine if this is due to bacterial growth during the incubation period.
If your broth looks cloudy, discard it and make another broth using less bacteria. Place the broth subcultures in an incubator at the temperature and time specified by your instructor. Separation of a mixed culture into individual colonies that can be subcultured to make pure cultures depends on how well the streak plate is prepared.
The goal of streak plate method is to dilute the cells by spreading them out over the surface of the agar. This is accomplished in stages, as will be demonstrated in lab before you try it yourself. Use the simulated agar surface below to practice the streak pattern using a pen or pencil. Also write M. As demonstrated, use a sterilized inoculating loop to pick up one M. Spread the bacteria over approximately a quarter of the plate, edge to edge. Consider this step 1. Flame the loop and cool it in the agar.
Overlap the step 1 streak times to pull out a reduced number of bacteria, and spread them out down the side of the plate. Consider this step 2. Overlap the step 2 streak times and spread over the surface. Continue this process, flaming the loop in between each step, until the entire surface of the agar plate is covered. After performing this with the M. Place the streak plate subcultures in an incubator at the temperature and time specified by your instructor.
Obtain one slant tube containing TSA, and label it using a small piece of tape with your name and culture name M. Using a sterilized inoculating loop , pick up a bacterial colony or piece of a colony from the surface of the plate culture of M.
Place the slant subculture in an incubator at the temperature and time specified by your instructor. Obtain one stab tube containing semisolid TSA, and label it using a small piece of tape with your name and culture name E. Using a sterilized inoculating needle , pick up a bacterial colony or piece of a colony from the surface of the plate culture of E. Withdraw the needle carefully and try to remove it by following the same stab line that you made pushing the needle down. Place the stab subculture in an incubator at the temperature and time specified by your instructor.
As you will learn, bacteria have preferred growth temperatures where their reproduction rate is the greatest. This lab is equipped with incubators set at either temperature. How long you plan to leave your cultures in an incubator should also be a consideration. Article Summary: Broth is a nutrient-infused liquid medium used for growing bacteria. Here is a summary of the advantages and disadvantages of this type of bacterial growth medium. Sterile liquid broth media in test tubes.
Media are initially sterilized, then inoculated with the bacteria of interest, and finally incubated for twenty four hours or more, to encourage bacterial growth. See Page 2 for more photos of different types of bacteria growing in liquid media! Broth and Agar Bacterial Growth Media. The two main types of bacterial growth media used are liquid broth and solid , Jell-o-like agar.
Each has specific advantages and disadvantages. Two types of media with similar implying names but very different functions, referred to as selective and differential media, are defined as follows.
Selective media are used for the growth of only selected microorganisms. Media lacking an amino acid such as proline in conjunction with E. Selective growth media are also used in cell culture to ensure the survival or proliferation of cells with certain properties, such as antibiotic resistance or the ability to synthesize a certain metabolite.
Normally, the presence of a specific gene or an allele of a gene confers upon the cell the ability to grow in the selective medium. In such cases, the gene is termed a marker. Selective growth media for eukaryotic cells commonly contain neomycin to select cells that have been successfully transfected with a plasmid carrying the neomycin resistance gene as a marker.
Gancyclovir is an exception to the rule as it is used to specifically kill cells that carry its respective marker, the Herpes simplex virus thymidine kinase HSV TK. Some examples of selective media include:. Non-selective versus selective media. While the plate on the right selectively only allows the bacteria Neisseria gonorrhoeae, to grow white dots.
Differential media or indicator media distinguish one microorganism type from another growing on the same media. This type of media is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria. Examples of differential media include:. Microbiologists rely on aseptic technique, dilution, colony streaking and spread plates for day-to-day experiments. Microbiologists have many tools, but four relatively simple techniques are used by microbiologists daily, these are outlined here.
Aseptic technique or sterile technique is used to avoid contamination of sterile media and equipment during cell culture. This technique involves using flame to kill contaminating organisms, and a general mode of operation that minimizes exposure of sterile media and equipment to contaminants. Serial Dilution : Example of Serial dilution of bacteria in five steps. The diluted bacteria were then spread plated.
When working with cultures of living organisms, it is extremely important to maintain the environments in which cells are cultured and manipulated as free of other organisms as possible.
This means passing rims and lids through the flame produced by a Bunsen burner in order to kill microorganisms coming in contact with those surfaces. Sterile technique, in general, is a learned state-of-being, or mantra, where every utilization of any sterile material comes with the caveat of taking every precaution to ensure it remains as free of contaminants as possible for as long as possible. A serial dilution is the step-wise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion.
A ten-fold serial dilution could be 1 M, 0. A culture of microbes can be diluted in the same fashion. For a ten-fold dilution on a 1 mL scale, vials are filled with microliters of water or media, and microliters of the stock microbial solution are serially transferred, with thorough mixing after every dilution step. The dilution of microbes is very important to get to microbes diluted enough to count on a spread plate described later.
Streak plate : Four streak plates. Successful streaks lead to individual colonies of microbes. In microbiology, streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria.
Samples can then be taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested. The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. This is dipped in an inoculum such as a broth or patient specimen containing many species of bacteria.
The sample is spread across one quadrant of a petri dish containing a growth medium, usually an agar plate which has been sterilized in an autoclave. Choice of which growth medium is used depends on which microorganism is being cultured, or selected for. Growth media are usually forms of agar, a gelatinous substance derived from seaweed.
Spread plates are simply microbes spread on a media plate. Microbes are in a solution, and can be diluted. They are then transferred to a petri dish with media specific for the growth of the microbe of interest. The solution is then spread uniformly through a number of possible means, the most popular is the use of sterile glass beads that are shook on top of the media, spreading the microbe-containing liquid evenly on the plate.
Also common is the use of a bent-glass rod, often referred to as a hockey stick, due to its similar shape. Spectrum of microbial keratitis in South Florida. Am J Ophthalmol ; 90 1 : 38— Single culture media in infectious keratitis. Cornea ; 18 3 : — The role of smears, cultures, and antibiotic sensitivity testing in the management of suspected infectious keratitis. Ophthalmology ; 1 : 23— Investigation of microbial keratitis: an audit from — Acta Ophthalmol Scand ; 74 2 : — Experience with a broth culture technique for diagnosis of bacterial keratitis.
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Am J Ophthalmol ; 88 3 Part 2 : — Comparison of techniques for culturing corneal ulcers. Ophthalmology ; 99 5 : — Morlet N, Daniell M. Microbial keratitis: what's the preferred initial therapy? View 2: empirical fluoroquinolone therapy is sufficient initial treatment.
Br J Ophthalmol. Bacteriological profile of ophthalmic infections in an Israeli hospital. Eur J Ophthalmol ; 9 2 : — J Clin Microbiol ; 39 12 : — Pantoea agglomerans as a cause of septic arthritis after palm tree thorn injury; case report and literature review. Arch Dis Child ; 88 6 : — Download references. You can also search for this author in PubMed Google Scholar. Correspondence to A Kratz. The authors have no proprietary interest in any of the materials or techniques used in this study.
Reprints and Permissions. Kratz, A. Broth cultures yield vs traditional approach in the workup of infectious keratitis. Eye 20, — Download citation. Received : 17 August Accepted : 05 January Published : 18 March
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