Plant Cell, Tissue and Organ Culture 52: 8388, 1998. 1998 Kluwer Academic Publishers.

. Printed in the Netherlands.

83

Laboratory contamination management: the requirement for microbiological quality assurance

Carlo Leifert1 & Stephen Woodward2
1 Department of Plant and Soil Science; 2 Department of

Forestry, University of Aberdeen, Aberdeen, Scotland, UK

Introduction Microbial contamination is the single most important cause of losses in commercial and scientic plant tissue culture laboratories [1,2]. However, even in commercial companies the severity and implications of the problem are not recognised or admitted. Many scientic laboratories fail to record contamination losses, and the micropropagation industry often only recognises the sources of contamination after severe losses have occurred. Following a rapid increase in the production of micropropagated plants in the 1980s [3], there has been a steady decline in the number of micropropagation laboratories in the 1990s, which was at least partially caused by the inability of laboratories to reduce contamination losses to a level which allows a predictable production output and quality of micropropagated plants. A level of contamination losses of not above 2% per subculture is usually seen as the minimum required to guarantee successful production. This level cannot be obtained without the application of regular quality assurance practices and the introduction of a microbiological production control strategy. Such practices are common practice in other industries affected by microbial contamination (e.g. food processing and pharmaceutical companies) [4]. Management/quality assurance strategies such as HACCP (Hazard Analysis Critical Control Points) can easily be applied to tissue culture laboratories. In micropropagation companies which have invested in the establishment of HACCP the additional costs were more than compensated for by the reduction in product losses and quality [1]. The microbial hazards in micropropagation can affect the tissue culture process itself, plant survival during weaning (if plants with fungal contaminants such as Botrytis cinerea are transferred to the nursery) and the customer of micropropagation laboratories (if pathogenic bacteria or viruses remain latent until plants have been sold) [510]. One important prereq-

uisite for the introduction of HACCP is a production recording system which allows the history of all plants to be traced in detail (including information on the stock plant, media batches, operator(s), ow cabinet(s) and growth room(s) plants have been exposed to). The establishment of HACCP then requires in-depth knowledge of (i) the identity and sources of potential hazards associated with specic stages of the production and distribution process (this will allow routine quality assurance based on the isolation and identication of indicator micro-organisms), (ii) methods for the early detection of hazards and (iii) the development of methods for the treatment of microbiological hazards. Since the rst workshop on bacteria and bacterialike contaminants in plant tissue cultures was held in Cork 8 years ago [11] considerable advances have been made in both the knowledge and methodology required to establish microbiological quality assurance systems for plant tissue-culture laboratories [1,2,12,13]. This review will critically evaluate recent published literature and personal experiences with the establishment of HACCP procedures in commercial micropropagation.

Identity and sources of potential hazards Contaminants described to cause severe economic losses in plant tissue culture laboratories include mites [1,14], thrips [1,14], fungi [1,1518], yeasts [1,19], bacteria [1,11,12,2022] and viruses [9,10]. Identication of the most common contaminants is relatively simple. The most common fungal and yeast contaminants can easily be identied by macroscopic examination of infected plant tissue cultures [7]. The mycelium/spore characteristics can be used for identication of important fungal/yeast genera associated with specic contamination sources (Table 1). It is also possible to separate the yeasts (yeasts grow vig-

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Table 1. Contaminants indicating specic contamination sources. Contaminant Bacteria Gram negatives Gram positives Gram-positive cocci (Staphylococcus epidermidis) Bacillus spp. Likely source for contamination Comments

Inefcient disinfection of explants Inefcient laboratory procedures Poor aseptic techniques in: (i) subculturing plants (ii) pouring of media Inefcient sterilisation of media

often Pseudomonas and Enterobacteriaceae Staphylococcus spp. are considered obligate inhabitants of animals often Bacillus subtilis and Bacillus pumilus Bacillus circulans can survive in 70% alcohol

Inefcient sterilisation of instruments used for subculture Fungi/yeasts General increase

Growthroom mite or thrip infestation

Especially when plantspecic growth rooms are affected Fusarium poe forms a white mycelium with a pink base Penicillium and pink yeast are very common air contaminants within buildings

Fusarium poe

Growth room infestation with Sideroptes graminis mites

Grey, black and green moulds (Botrytis, Aspergillus, Alternaria Penicillium spp.) Rhodotorula spp. (pink yeasts) Black moulds

High laboratory air contamination Faulty ow cabinets

Poor hygiene in growth rooms and plant and media cold stores

Moulds are often found in damp areas of the laboratory walls or grow in areas with frequent condensation. Spores are air transmitted Cladosporium spores are very common in outside air

Cladosporium spp.

Insufcient protection of laboratory against the outside air

85 orously and form white, off-white or red/pink nontranslucent growth, and produce a typical yeast odour in the culture container) from bacterial contaminants (bacteria usually produce only very slight growth on tissue culture media or, when more vigorous growth is produced, colonies are slimy and translucent in appearance) based on macroscopic assessment. Very few bacterial species produce characteristic symptoms or growth in plant tissue cultures [20,21] and different bacterial genera, therefore, cannot be separated based on their colony morphology. However, the main genera/groups of bacterial contaminants can be identied with just a few bacteriological tests and there are several test kit systems available now which can be used to conrm the genus and to identify bacterial contaminants to genus/species level (see Table 2). Genus level identication (which usually does not require the use of relatively expensive test kits) is often sufcient to identify the sources of bacterial contamination. Indicator contaminants which pinpoint specic contamination sources are described in Table 1. The API identication system (bioMrieux sa, 69280 Marcy-lEtoile, France) requires a range of six standard tests to be performed to select the appropriate test-strip (Table 1). The BIOLOG system (Biolog Inc. 3938 Trust Way, Hayward CA 94545, USA), which is based on standard microwell plates, only requires Gram staining of isolates in order to select a Gram-negative, Grampositive or yeast-specic identication system. Yeast and bacterial contaminants may also be identied by fatty acid proling [11] and or molecular methods such as plasmid proling and genetic ngerprinting [23]. latent infected material can cause severe losses at later stages of tissue culture or after weaning of plants [5,6,9,10,26,27]. Detection of latent bacteria is usually based on indexing of plant tissues [1]. This involves the transfer of pieces of plant material into solid or liquid bacteriological media after disinfection. The media used contain meat, yeast or plant extracts and have been described for sterility testing in other areas, such as the food and water industry and medical microbiology [2830]. Several indexing media which were developed specically for the detection of plant tissue-culture contaminants (e.g. medium 523 [32], George and Falkinghams mycobacterium detection medium TT [33] and Leifert and Waites Sterility Test Medium[1]) can now be obtained as commercial products ([31]; Sigma-Aldrich Company Ltd., Fancy Rd., Poole, Dorset BH17 7NH, UK). The advantage of using indexing media is that they allow growth and detection of a wide range of different bacterial contaminants [1] and many bacteria are detected even when present in very low numbers (101 to 102). However, different bacterial species show different amounts of growth on a particular medium (Table 3) and no bacteriological medium is able to detect all important contaminants [1,13]. Other limitations of indexing media have been described in detail elsewhere [1]. Commercial serological test kits are available for a wide range of plant viruses and specic plant pathogenic bacteria (e.g. Xanthomonas pelargonii, Clavibacter michiganense, Pseudomonas solanacearum, Erwinia amylovora, various pathovars of Erwinia syringae and Xanthomonas campestris and Erwinia carotovora pv. atroseptica) which may also stay latent in vitro ([31]; Adgen Plant Disease Diagnostics, Watson Peat Building, Auchincruive, Ayr KA6 5 HW, Scotland UK.; LOEWE Biochemica GmbH, Nordring 38, Postbox 9, 8156 Otterng bei Munchen, Germany). Such tests can be very sensitive, but are expensive and only detect one species of bacterium or virus. The suppression of many bacteria and viruses by plant resistance mechanisms and residues of the disinfectants used can result in low inocula being present in the plant pieces tested. Both indexing and serological tests should, therefore, be repeated both in vitro and after weaning of plants. Because of the relatively high cost of serological tests, these should only be used to check for organisms which are known to be a problem to particular plant species.

Detection of antagonists One of the most important sources of contamination is the explant during initiation of plant tissue culture [1,2,24]. The surface disinfection of the explant prior to its placement into a tissue culture test-tube or container is often inefcient. This problem may be due to the disinfectant being inactive or to micro-organisms being protected within the plant tissue used as the explant [11]. Fungal and yeast contaminants are easy to detect when they survive disinfection because they rapidly grow on plant tissue culture media [1]. Bacteria and viral contaminants, on the other hand, may not produce visible growth in the tissue culture medium [9,10,11,25]. Nevertheless, the propagation of such

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Table 2. Identication methods for bacterial contaminants. Classic bacteriological tests used CAT OF/F MOB GRAM COCC SPOR Suspected genus Staphyloc. Microc. Streptoc. Bacillus Bacillus Bacillus Bacillus corynef. corynef. corynef. Lactob. Acinetob. Vibrionac. Actinob. Pseu./Alca Flavob. Enterob. Enterob. Yeasts API Biolog

+ + + + + + + + + + + + + + + +

+ + + + + + + + + + + +

+ + + + + + + +

+ + + + + + + + + + + +

+ + + + oval

+ + + +

STAPH STAPH STREP 50CHB 50CHB 50CHB 50CHB Coryne Coryne Coryne 50CHL 20NE 20NE 20NE 20NE 20NE 20E 20E ID32C

GP GP GP GP GP GP GP GP GP GP GN GN GN GN GN GN GN GN YT

CAT, catalase; OF/F, anaerobic growth; MOT, motility; GRAM, Gram stain; COCC, coccus shaped as opposed to rod shaped; SPOR, heat-resistant spores formed; Staphyloc., Staphylococcus spp.; Microc., Micrococcus spp.; corynef., coryneform bacteria; Lactob., Lactobacillus spp.; Acinetob., Acinetobacter spp.; Vibrionac., Vibrionaceae; Actinob., Actinobacter spp.; Pseu., Pseudomonas spp.; Flavob., Flavobacterium spp.; Enterob., Enterobacteriaceae.

It is important to recognise that latent bacterial contaminants may also be introduced in the laboratory. Indexing plants at the initiation stage may, therefore, not prevent the accumulation of latent contamination when tissue cultures are continuously subcultured. To avoid this problem some laboratories maintain stock cultures of all their production plant lines. These are regularly tested for the presence of latent bacteria (every 24 subcultures/months) and index-positive cultures (those showing bacterial growth on bacteriological media) are discarded. Excesses of these stock cultures are regularly used to supplement the production cultures and only remain in production for a specic length of time (up to 2 years). Such production systems have proved to increase reliability,

and although they result in additional quality assurance cost, this is usually more than compensated by increased growth rates and lower contamination losses. The factors triggering latent bacteria to become virulent have previously been discussed in detail [1].

Conclusions Since the last symposium, our knowledge about the sources of contamination has increased considerably and more diagnostic tools are now available. However, we also had to realise that treatment of contamination (for example with antibiotics) is extremely

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Table 3. Growth (E625 ) of contaminants on Leifert & Waites medium and tryptone soya broth at different concentrations of nutrients. Test organism Leifert & Waites medium FS HS 1/10S 0.4 0.2

Tryptone soya broth FS HS 1/10S 0.4 0.3 0.2 0.5 0.7 0.2 0.8 0.4 0.4 0.4 0.8 0.5 0.2 0.4 0.3 0.2 0.4 0.6 0.3 0.7 0.3 0.4 0.5 0.8 0.2 0.2 0.3

Bacillus subtilis Cot1 Bacillus circulans A11 Clavibacter michiganense CM1 coryneform H416/3 Lactobacillus plantarum H260/6 Micrococcus kristinae Ho506/4 Staphylococcus saprophyticus Ch104/5 Acinetobacter calcoaceticus Ho295/35 Agrobacterium radiobacter Ger840/12 Agrobacterium tumefaciens As001/1 Pseudomonas maltophilia Del603/20 Xanthomonas campestris pv. campestris XCCi Xanthomonas campestris pv. vesicatoria XCV

0.4 0.2 0.2

0.3

0.3 0.2 0.7 0.2 0.3 0.3 0.8 0.8 0.4 0.3 0.3

0.8

0.8 0.2 0.8 0.4 0.9 0.8 0.6 0.7 0.4

0.3 0.2 0.2 0.2

0.8 0.5 0.9 0.8 0.7 0.5 0.3

0.4 0.4 0.7 0.5 0.2

FS, recommended concentration; HS, half the recommended concentration; 1/10S, one-tenth of the recommended concentration. Turbidity not detectable by visual assessment (E625 <0.2).

difcult and, with many contaminants, impossible [1,13,34,35]. This means even more emphasis will have to be placed on early detection and prevention of contamination at source. Due to the wide range of sources of contamination in a tissue-culture laboratory it is essential to introduce appropriate quality assurance systems such as HACCP which cover every potential source of contamination. This is not only essential to guarantee reliable production and quality of tissue cultured plants, but also operator safety. Contaminants which can cause disease such as ringworm (Trichophyton spp.), oral thrush (Candida albicans), gastroenteritis (Staphylococcus aureus) can be found in tissue cultures and at least one case where operators became infected from handling Trichophyton-infected tissue cultures has been reported [15]. Such systems have now been designed and become common practice in many commercial laboratories. The challenge for the next 8 years should be to perfect such quality assurance systems.

References
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