Why do microbiology labs need stock cultures?
There are a number of reasons why a microbiology laboratory needs stock cultures in good condition. The typical stock culture collection may contain isolates that fall into one or more of the following categories: Isolates used in the preparation of inoculated samples and specimens for quality control and training purposes
What is the most important task in a microbiology lab?
One of the most important, yet often neglected, tasks in any routine microbiology laboratory is to maintain a collection of bacterial and fungal stock cultures.
What is a stock solution?
making dilutions working with stock solutions Working with Stock Solutions We define a stock solutionas a concentrate, that is, a solution to be diluted to some lower concentration for actual use.
What is the purpose of the microbiology safety manual?
This manual explains the basic techniques necessary to carry out microbiology experiments safely and effectively, and is intended as a guide for teachers and technicians.
What is working stock culture?
Working Stock Cultures: A working stock culture is growth derived from a reference stock culture. Guidelines and standards outline how working stock cultures must be processed and how often they can be subcultured. Subcultures (Passages): A subculture is simply the transfer of established microorganism growth on.
What is a stock culture in microbiology?
[ stŏk ] n. A culture of a microorganism maintained solely to keep it viable for subculture into fresh medium.
Why is stock culture important?
Stock cultures of microorganisms kept in a laboratory provide the organisms required for conducting experiments (Fig. S1). As such, the stock cultures are extremely important resources, and should be maintained in a manner that ensures their long-term persistence.
How does Microbiology maintain stock culture?
Frozen storage on cryoprotectant beads Perhaps the most practical method of long term microbial stock culture storage for smaller laboratories and those engaged in routine testing is to freeze cultures onto porous beads designed to allow cells and spores to attach to their surface – providing some degree of protection ...
How do you make bacteria stock?
Inoculate 5 ml LB with a single colony from an overnight plate or with 5 µl of mini-prep DNA bacterial culture and grow overnight.Add 800 µl of the fresh overnight culture + 200 µl 75% glycerol in a clean 1.5 ml Eppendorf tube; mix thoroughly with pipette.Transfer to Nunc tube, and store at -70°C.
How is the reserve culture used differently from the working stock culture?
The working stock culture is used for all subcultures, staining, biochemical testing whereas the reserve stock culture is stored in the refrigerator and used only if the working stock culture becomes contaminated.
How do you maintain stock cultures?
Preserving Stock Cultures A popular method for preserving microorganisms is through lyophilization, or freeze drying. This method is best for long term storage. The lyophilization process requires costly equipment including a lyophilizer, vacuum pump, generator, etc.
What is the importance of the preservation of bacterial stock culture?
The goal of preserving the cultures is to slow that death rate so that when the culture is revisited, some of the cells are still viable and available for culturing. The reasons the cells die can be numerous, but in every instance are based on the inherent chemistry of the cells and their environment.
How do you store microorganisms?
For long term storage, microorganisms are cryopreserved at temperatures below freezing (-20°C, -80°C, or liquid nitrogen) or freeze dried (lyophilized). Cryopreservation is suitable for a wide range of bacteria, algae, fungi, viruses, and protozoa.
What medium is used for preparation of stock cultures?
Agar slants are commonly used to maintain stock cultures because they provide a fairly broad surface for growth and observation, but do not dry out as fast as agar plates. c. Streak plates - Molten agar media poured into Petri plates will cool to form broad, thin slabs of solid media known as streak plates.
How do you maintain anaerobic stock cultures?
Store at room temperature in a candle jar or in a container with a CO2 packet. Store at room temperature. Store in anaerobic conditions at room temperature. Store on chocolate agar at 35°C in microaerophilic conditions.
How do you store bacterial cultures?
Working bacterial stocks can be streaked onto agar plates and stored at 4°C for daily or weekly use. Culture dishes should be wrapped with laboratory sealing film (plastic or paraffin) and stored upside down (agar side up) to minimize contamination and to keep both the culture and agar properly hydrated.
What is labelling mistakes?
Labelling mistakes – subculturing a large number of agar slants many times introduces a significant chance of a culture being wrongly labelled
How are stock cultures stored?
After incubation at a suitable temperature, the subcultures are stored in a refrigerator until required, or until the next subculturing is due. This system works up to a point, but it does have some serious drawbacks.
What is a cryoprotectant bead?
Cryoprotectant beads allow routine labs to maintain a stock culture collection simply and at low cost.
How to prepare a colonial growth?
Outline method 1. Prepare a suspension of cells or spores of the isolate to be stored, preferably using an 18 to 24 hour old culture grown on solid medium. The colonial growth is suspended in a cryoprotectant fluid, such as Brucella Broth with Glycerol, in a cryogenic vial containing 20-30 of the porous beads. 2.
What temperature is liquid nitrogen?
The temperature of liquid nitrogen is -196 o C, well below the temperature at which all metabolic activity is thought to cease. As with freeze-drying, not all microbial cultures will survive the process, but those that do may survive for many years. Indeed viability for some isolates may be better than for freeze-dried cultures. However, cryogenic storage is expensive, requires significant amounts of liquid nitrogen and is probably only suitable for larger reference laboratories with extensive culture collections.
How many passages of culture should be made from the original strain?
In fact, the American Type Culture Collection (ATCC) recommends that no more than five passages (subcultures) should be made from the original type strain, with the first passage being defined as the culture prepared from the vial supplied by ATCC.
How many passages of stock culture should be made?
In fact, the American Type Culture Collection (ATCC) recommends that no more than five passages (subcultures) should be made from the original type strain, with the first passage being defined as the culture prepared from the vial supplied by ATCC.
How often were S. aureus cultures subcultured?
Genetic drift is also a possibility, but it is not known how often the cultures were subcultured. The authors learned, however, one of the eight laboratories had purchased the S. aureus strain eight to ten years earlier and three of the laboratories had no information about when the S. aureus strains were purchased.
How long can you keep a culture on agar?
Several industry standards recommend discarding working stock cultures after one month: ISO 11133 says cultures can be stored on agar at a suitable temperature for up to four weeks. CLSI recommends maintaining strains used in antimicrobial susceptibility tests for no more than a month on agar.
How long can antibiotic resistant strains be kept on agar?
CLSI cautions some antibiotic resistant strains should only be maintained on agar for two weeks because these strains can easily lose the plasmid containing the antimicrobial properties. 8. 5. No support when times get tough. Microbiologics issues Certificates of Analysis for every lot it produces.
How long can bacteria be stored in a freezer?
Bacteria stored for one year in a general-purpose freezer at -20˚C remain viable for about one year before ice crystals and chemical reactions within the cell cause damage. 2,3 Several industry standards recommend microorganisms be stored in a cryoprotectant in an ultra-low freezer of -60˚C or colder because lower temperatures result in longer viability of the cell.
Why is genetic modification stored in a freezer?
Genetic modification due to storage in a freezer too warm to adequately protect the microorganisms
How does subculture affect mutation?
4. Excessive subcultures increase the risk of mutation. By using Microbiologics cultures and following its plan for maintaining cultures for one month, laboratories can limit the number of passages from the reference stock culture. According to the United States Pharmacopeia (USP), excessive subculturing can increase the risk ...
What temperature should reference stock be stored at?
ISO 11133 says, “Reference stocks should be stored in multiple portions, usually either deep-frozen, e.g. below -70˚C, or lyophilized. At a higher temperature, duration of viability might be reduced and genetic modification might occur.” 5
What is microbiology in schools?
Microbiology is a popular option for practical work in schools. This manual, which explains the basic techniques necessary to carry out microbiology experiments safely and effectively, is intended as a guide for teachers and technicians. The Microbiology Society, in association with the Microbiology in Schools Advisory Committee (MiSAC) has also produced a book of 21 practical investigations which complements the manual – Practical Microbiology for Secondary Schoolsis suitable for use with Key Stages 3, 4 and post-16 and the equivalent Scottish qualifications. This resource can be downloaded from the Microbiology Society’s dedicated education website: www.microbiologyonline.org.uk The Microbiology Society offers schools membership and runs an advice service on microbiology teaching. For further details, email [email protected], telephone +44 (0)20 7685 2682 or write to the address at the bottom of the page. Other useful sources of advice: x CLEAPSS – www.cleapss.org.uk The Gardiner Building, Brunel Science Park, Uxbridge UB8 3PQ x Microbiology in Schools Advisory Committee (MiSAC) – www.misac.org.uk c/o NCBE (see address below)
What is GMLP training?
Training in GMLP is aimed at developing proficiency in containing any uncontrolled spread of microbes in order to protect: x practical investigations from becoming contaminated with microbes from external sources x the operators (students, teachers and tech- nicians) from the very small possibility of infection. (The teacher superising the practical session ust ae theseles aare of any edical condition that could cause the student to be at greater ris than aerage in the laboratory e.g. treatent ith iunosuppressie drugs etc.)
What is the risk of a spillage?
Spillages also carry a risk of generatingaerosols (an invisible ‘mist’ of small dropletsof moisture ) which may contain microbes andmight be inhaled. The risk of spillages occurring is lessened by using cultures grownon agar instead of in liquid media wheneverpossible. Care should also be taken to avoidgenerating aerosols during practical work. Therisk is minimised by adhering to GMLP withspecial attention to the correct use of pipettes(see Inoculation and other aseptic procedurespage 8).
Why do you need a young culture?
Always use a young culture because older cultures of Gram-positive bacteria tend to lose the ability to retain the crystal violetiodine complex and appear to be Gram-negative but some bacteria are naturally only weakly Gram-positive.
How are wire loops sterilised?
Wire loops are sterilised using red heat in a Bunsen flame before and after use . They must be heated to red hot to makesure that any contaminating bacterial spores are destroyed. The handle of the wire loop is held close to the top, as youwould a pen, at an angle that is almost vertical. This leaves the little finger free to take hold of the cotton woolplug/screw cap of a test tube/bottle.
Why are plugs made of cotton wool?
Plugs made of non-absorbentcotton wool are used in test tubes and pipettes to prevent micro-organisms from passing in or out and contaminating either the culture or the environment. The necessary movements of air in andgaseous products out are not prevented and the gaps between the cotton wool fibres are even wide enough for micro-organisms to pass through. However, this does not happen because micro-organisms (negatively charged) are ‘filtered’ out by being attracted to and adsorbed on the oppositely charged cotton wool. The cotton wool must remain drybecause this filtration property is lost if the cotton wool becomes moist – hence the use of non-absorbentcotton wool.
How to dispose of broken glass?
It should be swept carefully into a suitable container, autoclaved and disposed of in a puncture proof container.