Preservation and Storage Preservation and Storage methods of Fungal Culturemethods of Fungal Culture

Marlia Singgih WibowoSchool of Pharmacy

Institut Teknologi Bandung

OutlineOutline

General PrinciplesPreservation techniqueAdvantage and DisadvantagesSelection of method

General PrincipleGeneral Principle

The aim is to preserve fungal culture is to maintain fungal strain in a media without any changes in their morphology, physiology, or genom

General PrincipleGeneral Principle

Microorganisms can be originated from many sourcesExample : Penicilliumdigitatum

General PrincipleGeneral PrinciplePreservation techniques are various, can be done by reducing rate of metabolism or prevent the metabolismGood preservation can be provided if the culture is in good condition and at their optimum growthMicroorganisms grow in different environment and conditionsSome of them have certain specificity for their growth

General PrincipleGeneral Principle

In general, Microorganisms can grow well in the media which is made very similar to their original habitat, example : soil, part of plants, or water component, etc.

Growth of MicroorganismsGrowth of MicroorganismsGrowth Media can be vary, for example Raper & Thom (1949) used Czapek's Agar, Steep Agar and Malt Extract Agar for Penicillia dan AspergilliPitt (1980) recommends Czapek Yeast Autolysate (CYA) and Malt Extract Agar (MEA) for penicilliaStandarization of media formula is very important Media will influence morphology and colony color, because composition of media will influence the synthesis of certain metabolites or induce a specific properties from the microorganisms.

Growth Growth The influence of media composition on colony morphology

Contamination on fungal cultureContamination on fungal cultureFungal culture is often contaminated by Tyroglyphus or TarsonemusIn Nature, they are abundance in soil, and in almost organic materialThe contaminant is brought into laboratory from plants, expired products, on shoes, insects or other microbial cultures Fast growing in humid condition at room temperatureFungal culture can be ruin and cannot be preserved

How to avoid ContaminationHow to avoid ContaminationDo the work according to SOP Keep clean and keep healthy Always check every material/substances entering the laboratoryCover all cultures according to SOP

Methods to avoid contamination Methods to avoid contamination Hygiene

Clean up all surface of benches and avoid the culture from exposure to dust and open air .Wash all glassware and apparatus using desinfectant.

FumigationIt is used in between chemical sterilisation for laboratory area or corridor. The work should be done by the authority person.

Mechanical and chemical barriersUniversal bottles, cottons, plugged tubes

Place the culture safely in proper rack Use 4-8°C refrigeratorUse “deep freezer” (< -20°C)Pour mineral oil over the culturePlace silica gel around the culture tubesUse Freeze-dry methodUse ultra-low temperatures case

Methods to avoid contamination Methods to avoid contamination

Preservation and StoragePreservation and StorageContinuous culture

On solid media or liquid mediaRefrigerationUnder mineral oilUnder water

Heat Drying Freeze dryingCryopreservation

Preservation and Storage Preservation and Storage

Culture sample divided into 3 cultures :1 culture for seed stock1 culture for working culture1 culture for retain culture After presrvation, viability, purity, and identity should be checked and compared to original data or reference dataMorphology, pathogenity, phyisical and biochemical properties should be tested Every data should be documented and kept for future needs.

Microorganisms Preservation MethodMicroorganisms Preservation Method

1. Growing on agar slant– Unexpensive & easy– Store in refrigerator

can reduce the frequency of sub-culturing

Disadvantages of sub-culturing method

• Possibility of genetic variation due to repeated transfer, lack of pathogenicity, or morphology and physiology

• Possibility of contaminations is very high • Need extra attention to control the culture to

avoid mix-up or contamination.

The advantage of sub-culture method on agar :

Cultures can be stored for certain period of time, under supervision of the competent personel. Culture collection can be stored by an expert Koleksi biakan dapat disimpan dalam waktucukup lama dibawah pengawasan ahlinya.The method is unexpensive and no need special treatment. Ideal for small laboratoriesThe Growth is easy due to short adaptation process : 2 – 4 weeks or 2 – 4 months

Under mineral oil Under mineral oil

Cultures on agar slant in 30 ml of universal bottles , then mineral oil is poured onto the cultures. This is to avoid dehydration and to slow down metabolism activities and growth, due to lack of oxygen

2. Preservation using 2. Preservation using Mineral OilMineral Oil

First introduced by Buell & Weston (1947)Mineral oil used : sterile liquid paraffin or medicinal paraffin with specific gravity 0.830-0.890 Oil is sterilized using autoklave 2 times at 121°C for 15 minutesRe-growing from oil : withdraw the oil and take some colonies using sterile loop, inoculate on the middle of fresh agar

Disadvantages of mineral oil method :

Contamination of microbial spores from air Slow growing at retrievalGrowing under unpleasant oil, can induce mutation

Advantages of Mineral Oil Method :

Inexpensive and easy to operate Some microorganisms cannot grow in mineral oilConvenient for small laboratorium with limited facilities

3. Preservation in Water 3. Preservation in Water

Agar pieces or spores suspension can be stored in water

1. Agar piece about 6 mm cut from the edge of colony

2. Place in sterile water in McCartney Bottle and seal , store at 20-25°C.

3. Retrieve the colony by inoculate the culture upside down on fresh media

Example : Phytophthora dan Pythium , fungal cultures can be stored for 2-3 years, without any physical changes (Onions & Smith, 1984).

Procedure of water preservation

4. Drying and Freeze4. Drying and Freeze--dryingdrying

Water discharge can inhibit rate of cell metabolismCan be done by air drying Addition of absorbant , i.e. soil, silica gel, or other desiccant Freeze-dry with vacuum from freeze form by sublimation process

Response of cells to freezing. Slow cool: cells can maintaing osmosticequilibrium by water efflux; thus cells shrink and only extracellular ice forms. Rapid cool: cells cannot lose water rapidly enough to maintain osmotic equilibrium; thus cells shrink slightly and contain a few large

ice crystals. Very rapid cool: cells contain a large number of small ice crystals.

SLOWCOOL

RAPIDCOOL

VERYRAPIDCOOL

--22OOCC

--55OOCC

< < --1010OOCC

What happens to a cell What happens to a cell when it freezes?when it freezes?

5. Preservation in Silica Gel 5. Preservation in Silica Gel

Sporulating fungi can be stores for 7-18 years C.F. Roberts (1962) from University of Leicester, preserved fungi more than 25 years and proved stabile

Procedure of Silica Gel Preservation Procedure of Silica Gel Preservation

1. One third of 30 ml universal bottle filled with silica gel and sterilize by dry heat (180°C for 3 hrs).

2. Freeze the bottle at - 20°C3. Prepare spore suspension in 5%w/v of cold

skimmed milk. Add about 1 ml of culture into the silica gel and shake to homogenize it.

5. Store the bottle with the cap slightly loose for 10-14 days at 25°C until the silica gel crystalized.

6. Tighten up the cap and store the bottle at 4°C

Most fungi can survive with this method (Perkins, 1962; Ogata, 1962; Onions, 1977; Smith & Onions, 1983). Spores with thin cell wall cannot surviveThe culture for preservation should be healthy , otherwise it will show different morphology

Re Growing on agar after silica gel Re Growing on agar after silica gel preservationpreservation

Disadvantages of Silica GelDisadvantages of Silica Gel Method

Good for sporulating fungi. Pythium, Phytophthora and some Oomycota, poorly kept in SG.Contamination easily happened after many times retrieving.

Advantages of Silica Gel methodAdvantages of Silica Gel methodSimple and inexpensive Good for sporulating fungi , including Basidiomycota.Bacterial contamination can be avoided because of very dry conditionRetrieving process is very easy, however stock culture should be separated with working culture

6. Preservation in Soil6. Preservation in Soil

Soil should be autoclaved 2 times (121°C for 15 min)Inoculation with 1 ml of spores suspension in sterile waterIncubate at 20-25°C for 5-10 days or depends on fungal growing rate

Fusarium (Gordon, 1952; Booth, 1971)Atkinson (1953) keep Rhizopus, Alternaria, Aspergillus, Circinella and Penicillium. Septoria (Shearer et al., 1974) and Pseudocercosporella (Reinecke & Fokkema, 1979). Gordon's collection showed that 76% of Fusarium equiseti, 75% F. semitectum dan50% F. acuminatum grown uncontrolled (Booth, 1971). For Fusarium , preservation in soil is better than in oil.

Examples of soil preservationExamples of soil preservation

Can survive up to 10 tahun.Inexpensive and easy to operate

Advantages of Soil preservation Advantages of Soil preservation

Possibility of genetic variationSome fungi cannot be desiccatedPossibility of contamination is very high

Disadvantages of soil preservationDisadvantages of soil preservation

7. 7. CryopreservationCryopreservationLowering the temperature of biological material reduces the rate of metabolism until, when all internal water is frozen, no further biochemical activity Little metabolic activity takes place below -70°CRecrystallization of ice can occur at temperatures above -139°C (Morris, 1981) The shelf-life is often considered as infinity, animal cells may survive 32 000 years before a lethal background dosage of radiation is reached (Ashwood-Smith & Grant, 1976). Freezer storage at -80°C has been successfully used for the shorter term giving ca. 15 years storage (Yukio, 1987; Ito, 1991).

Cooling rateCooling rateProgrammable coolerFreezerSuspended in nitrogen vapourPlunging into liquid nitrogen

CryopreservationCryopreservation

In or above liquid nitrogen

CryopreservationCryopreservation

Advantages of liquid nitrogen storageWhen stored in sealed ampoules the fungi are free of contamination.The majority of both sporulating and non-sporulating fungi survive well, giving the method a greater range of successful application.Shelf-life is considered to be limitless if storage temperature is kept below -139°C.

Apparatus is costlySupply of liquid can prove expensive Supply failure may result in collection lossIn hotter climates evaporation rate fasterA regular supply of liquid cannot be obtained in some parts of the world Storage vessels must be kept in a well-ventilated roomThe double-jacketed, vacuum-sealed storage vessels can corrode and rupture. Such a failure may result in the loss of all the strains stored in it.

Disadvantages of liquid nitrogen Disadvantages of liquid nitrogen storagestorage

SummarySummaryMethod Cost Stability Longevity

Cryopreservation below –130oC

High Excellent >10 000 years

Freeze-drying High Little change reported

4-40 years

Silica gel Low Little change reported

5-20 years

Water Low Variation linked to storage time

2-7 years

Soil Low Variation common

5-20 years

Oil Low Change expected with time

1-50 years

Growth 18-22 oC Low-Medium Change expected with time

2 weeks to 6 months

Growth 4-7 oC Medium Change expected with time

6-12 months