Plant Cell, Tissue and Organ Culture52: 83–88, 1998.© 1998Kluwer Academic Publishers. Printed in the Netherlands.

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Laboratory contamination management: the requirement formicrobiological quality assurance

Carlo Leifert1 & Stephen Woodward21Department of Plant and Soil Science;2Department of Forestry, University of Aberdeen, Aberdeen, Scotland, UK

Introduction

Microbial contamination is the single most importantcause of losses in commercial and scientific plant tis-sue culture laboratories [1,2]. However, even in com-mercial companies the severity and implications of theproblem are not recognised or admitted. Many scien-tific laboratories fail to record contamination losses,and the micropropagation industry often only recog-nises the sources of contamination after severe losseshave occurred. Following a rapid increase in the pro-duction of micropropagated plants in the 1980s [3],there has been a steady decline in the number of mi-cropropagation laboratories in the 1990s, which wasat least partially caused by the inability of laborato-ries to reduce contamination losses to a level whichallows a predictable production output and quality ofmicropropagated plants.

A level of contamination losses of not above 2%per subculture is usually seen as the minimum requiredto guarantee successful production. This level cannotbe obtained without the application of regular qualityassurance practices and the introduction of a micro-biological production control strategy. Such practicesare common practice in other industries affected bymicrobial contamination (e.g. food processing andpharmaceutical companies) [4]. Management/qualityassurance strategies such as HACCP (Hazard AnalysisCritical Control Points) can easily be applied to tissueculture laboratories. In micropropagation companieswhich have invested in the establishment of HACCPthe additional costs were more than compensated forby the reduction in product losses and quality [1].

The microbial hazards in micropropagation can af-fect the tissue culture process itself, plant survivalduring weaning (if plants with fungal contaminantssuch asBotrytis cinereaare transferred to the nursery)and the customer of micropropagation laboratories(if pathogenic bacteria or viruses remain latent untilplants have been sold) [5–10]. One important prereq-

uisite for the introduction of HACCP is a productionrecording system which allows the history of all plantsto be traced in detail (including information on thestock plant, media batches, operator(s), flow cabinet(s)and growth room(s) plants have been exposed to).The establishment of HACCP then requires in-depthknowledge of (i) the identity and sources of potentialhazards associated with specific stages of the produc-tion and distribution process (this will allow routinequality assurance based on the isolation and identifi-cation of indicator micro-organisms), (ii) methods forthe early detection of hazards and (iii) the develop-ment of methods for the treatment of microbiologicalhazards.

Since the first workshop on bacteria and bacteria-like contaminants in plant tissue cultures was held inCork 8 years ago [11] considerable advances havebeen made in both the knowledge and methodol-ogy required to establish microbiological quality as-surance systems for plant tissue-culture laboratories[1,2,12,13]. This review will critically evaluate recentpublished literature and personal experiences with theestablishment of HACCP procedures in commercialmicropropagation.

Identity and sources of potential hazards

Contaminants described to cause severe economiclosses in plant tissue culture laboratories include mites[1,14], thrips [1,14], fungi [1,15–18], yeasts [1,19],bacteria [1,11,12,20–22] and viruses [9,10]. Identifi-cation of the most common contaminants is relativelysimple. The most common fungal and yeast conta-minants can easily be identified by macroscopic ex-amination of infected plant tissue cultures [7]. Themycelium/spore characteristics can be used for iden-tification of important fungal/yeast genera associatedwith specific contamination sources (Table 1). It isalso possible to separate the yeasts (yeasts grow vig-

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Table 1. Contaminants indicating specific contamination sources.

Contaminant Likely source for contamination Comments

Bacteria

Gram negatives Inefficient disinfection of explants oftenPseudomonas

Gram positives Inefficient laboratory procedures and Enterobacteriaceae

Gram-positive cocci Poor aseptic techniques in: Staphylococcusspp.

(Staphylococcus (i) subculturing plants are considered obligate

epidermidis) (ii) pouring of media inhabitants of animals

Bacillusspp. Inefficient sterilisation of media oftenBacillus subtilis

andBacillus pumilus

Inefficient sterilisation of Bacillus circulanscan

instruments used for subculture survive in 70% alcohol

Fungi/yeasts

General increase Growthroom mite or thrip Especially when plant-

infestation specific growth rooms are

affected

Fusarium poe Growth room infestation with Fusarium poeforms a

Sideroptes graminismites white mycelium with a

pink base

Grey, black and High laboratory air contamination Penicilliumand pink yeast

green moulds Faulty flow cabinets are very common air

(Botrytis, Aspergillus, contaminants within

buildings

Alternaria

Penicilliumspp.)

Rhodotorulaspp.

(pink yeasts)

Black moulds Poor hygiene in growth rooms Moulds are often found in

and plant and media cold stores damp areas of the

laboratory

walls or grow in areas with

frequent condensation.

Spores are air transmitted

Cladosporiumspp. Insufficient protection of laboratory Cladosporiumspores are

against the outside air very common in outside air

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orously and form white, off-white or red/pink non-translucent growth, and produce a typical yeast odourin the culture container) from bacterial contaminants(bacteria usually produce only very slight growth ontissue culture media or, when more vigorous growthis produced, colonies are slimy and translucent in ap-pearance) based on macroscopic assessment. Very fewbacterial species produce characteristic symptoms orgrowth in plant tissue cultures [20,21] and differentbacterial genera, therefore, cannot be separated basedon their colony morphology.

However, the main genera/groups of bacterial con-taminants can be identified with just a few bacteri-ological tests and there are several test kit systemsavailable now which can be used to confirm the genusand to identify bacterial contaminants to genus/specieslevel (see Table 2). Genus level identification (whichusually does not require the use of relatively expen-sive test kits) is often sufficient to identify the sourcesof bacterial contamination. ‘Indicator contaminants’which pinpoint specific contamination sources are de-scribed in Table 1. The API identification system(bioMérieux sa, 69280 Marcy-l’Etoile, France) re-quires a range of six standard tests to be performedto select the appropriate test-strip (Table 1).

The BIOLOG system (Biolog Inc. 3938 Trust Way,Hayward CA 94545, USA), which is based on stan-dard microwell plates, only requires Gram stainingof isolates in order to select a Gram-negative, Gram-positive or yeast-specific identification system. Yeastand bacterial contaminants may also be identified byfatty acid profiling [11] and or molecular methodssuch as plasmid profiling and genetic fingerprinting[23].

Detection of antagonists

One of the most important sources of contaminationis the explant during initiation of plant tissue culture[1,2,24]. The surface disinfection of the explant priorto its placement into a tissue culture test-tube or con-tainer is often inefficient. This problem may be due tothe disinfectant being inactive or to micro-organismsbeing protected within the plant tissue used as the ex-plant [11]. Fungal and yeast contaminants are easyto detect when they survive disinfection because theyrapidly grow on plant tissue culture media [1]. Bacte-ria and viral contaminants, on the other hand, may notproduce visible growth in the tissue culture medium[9,10,11,25]. Nevertheless, the propagation of such

‘latent’ infected material can cause severe losses atlater stages of tissue culture or after weaning of plants[5,6,9,10,26,27]. Detection of ‘latent’ bacteria is usu-ally based on ‘indexing’ of plant tissues [1]. Thisinvolves the transfer of pieces of plant material intosolid or liquid bacteriological media after disinfection.The media used contain meat, yeast or plant extractsand have been described for sterility testing in otherareas, such as the food and water industry and med-ical microbiology [28–30]. Several ‘indexing media’which were developed specifically for the detectionof plant tissue-culture contaminants (e.g. medium 523[32], George and Falkingham’s mycobacterium detec-tion medium TT [33] and Leifert and Waites SterilityTest Medium[1]) can now be obtained as commercialproducts ([31]; Sigma-Aldrich Company Ltd., FancyRd., Poole, Dorset BH17 7NH, UK).

The advantage of using indexing media is that theyallow growth and detection of a wide range of differ-ent bacterial contaminants [1] and many bacteria aredetected even when present in very low numbers (101to 102). However, different bacterial species showdifferent amounts of growth on a particular medium(Table 3) and no bacteriological medium is able todetect all important contaminants [1,13]. Other limita-tions of ‘indexing media’ have been described in detailelsewhere [1].

Commercial serological test kits are available for awide range of plant viruses and specific plant patho-genic bacteria (e.g.Xanthomonas pelargonii, Clav-ibacter michiganense, Pseudomonas solanacearum,Erwinia amylovora, various pathovars ofErwiniasyringaeand Xanthomonas campestrisand Erwiniacarotovorapv. atroseptica) which may also stay la-tent in vitro ([31]; Adgen Plant Disease Diagnostics,Watson Peat Building, Auchincruive, Ayr KA6 5HW, Scotland UK.; LOEWE Biochemica GmbH, Nor-dring 38, Postbox 9, 8156 Otterfing bei Munchen,Germany). Such tests can be very sensitive, but areexpensive and only detect one species of bacterium orvirus.

The suppression of many bacteria and viruses byplant resistance mechanisms and residues of the disin-fectants used can result in low inocula being present inthe plant pieces tested. Both indexing and serologicaltests should, therefore, be repeated bothin vitro andafter weaning of plants. Because of the relatively highcost of serological tests, these should only be used tocheck for organisms which are known to be a problemto particular plant species.

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Table 2. Identification methods for bacterial contaminants.

Classic bacteriological tests used

CAT OF/F MOB GRAM COCC SPOR Suspected API Biolog

genus

+ + – + + – Staphyloc. STAPH GP

+ – – + + – Microc. STAPH GP

– + – + + – Streptoc. STREP GP

+ + + + – + Bacillus 50CHB GP

+ – + + – + Bacillus 50CHB GP

+ + – + – + Bacillus 50CHB GP

+ – – + – + Bacillus 50CHB GP

+ + + + – – corynef. Coryne GP

+ – + + – – corynef. Coryne GP

+ + – + – – corynef. Coryne GP

– + – + – – Lactob. 50CHL GN

+ – – – + – Acinetob. 20NE GN

+ + + – – – Vibrionac. 20NE GN

+ + – – – – Actinob. 20NE GN

+ – + – – – Pseu./Alca 20NE GN

+ – – – – – Flavob. 20NE GN

+ + + – – – Enterob. 20E GN

+ + – – – – Enterob. 20E GN

+ + + oval – Yeasts ID32C YT

CAT, catalase; OF/F, anaerobic growth; MOT, motility; GRAM, Gram stain; COCC, coccusshaped as opposed to rod shaped; SPOR, heat-resistant spores formed;Staphyloc., Staphylococ-cusspp.;Microc., Micrococcusspp.; corynef., coryneform bacteria;Lactob., Lactobacillusspp.;Acinetob., Acinetobacterspp.; Vibrionac., Vibrionaceae;Actinob., Actinobacterspp.; Pseu.,Pseudomonasspp.;Flavob., Flavobacteriumspp.; Enterob., Enterobacteriaceae.

It is important to recognise that latent bacterialcontaminants may also be introduced in the laboratory.Indexing plants at the initiation stage may, therefore,not prevent the accumulation of ‘latent’ contaminationwhen tissue cultures are continuously subcultured. Toavoid this problem some laboratories maintain stockcultures of all their production plant lines. These areregularly tested for the presence of latent bacteria(every 2–4 subcultures/months) and ‘index-positive’cultures (those showing bacterial growth on bacteri-ological media) are discarded. Excesses of these stockcultures are regularly used to supplement the pro-duction cultures and only remain in production fora specific length of time (up to 2 years). Such pro-duction systems have proved to increase reliability,

and although they result in additional ‘quality assur-ance’ cost, this is usually more than compensatedby increased growth rates and lower contaminationlosses. The factors triggering ‘latent’ bacteria to be-come virulent have previously been discussed in detail[1].

Conclusions

Since the last symposium, our knowledge about thesources of contamination has increased considerablyand more diagnostic tools are now available. How-ever, we also had to realise that treatment of conta-mination (for example with antibiotics) is extremely

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Table 3. Growth (E625) of contaminants on Leifert & Waites medium and tryptonesoya broth at different concentrations of nutrients.

Test organism Leifert & Waites medium Tryptone soya broth

FS HS 1/10S FS HS 1/10S

Bacillus subtilisCot1 0.4 0.4 0.3 0.4 0.4 0.3

Bacillus circulansA11 0.2 0.2 ∗∗ 0.3 0.3 ∗∗Clavibacter michiganense ∗∗ 0.2 0.3 0.2 0.2 ∗∗CM1

coryneform H416/3 ∗∗ 0.2 0.5 0.4 0.3

Lactobacillus plantarum 0.8 0.8 0.7 0.7 0.6 0.2

H260/6

Micrococcus kristinae ∗∗ 0.2 0.2 0.2 0.3 0.2

Ho506/4

Staphylococcus 0.8 0.8 0.3 0.8 0.7 0.2

saprophyticusCh104/5

Acinetobacter calcoaceticus 0.5 0.4 0.3 0.4 0.3 ∗∗Ho295/35

Agrobacterium radiobacter 0.9 0.9 0.8 0.4 0.4 0.4

Ger840/12

Agrobacterium tumefaciens 0.8 0.8 0.8 0.4 0.5 0.4

As001/1

Pseudomonas maltophilia 0.7 0.6 0.4 0.8 0.8 0.7

Del603/20

Xanthomonas campestris 0.5 0.7 0.3 0.5 0.2 0.5

pv. campestris XCCi

Xanthomonas campestris 0.3 0.4 0.3 0.2 0.2 0.2

pv. vesicatoria XCV

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

difficult and, with many contaminants, impossible[1,13,34,35]. This means even more emphasis willhave to be placed on early detection and preventionof contamination at source. Due to the wide rangeof sources of contamination in a tissue-culture labo-ratory it is essential to introduce appropriate qualityassurance systems such as HACCP which cover everypotential source of contamination. This is not only es-sential to guarantee reliable production and quality oftissue cultured plants, but also operator safety. Con-taminants which can cause disease such as ringworm(Trichophytonspp.), oral thrush (Candida albicans),gastroenteritis (Staphylococcus aureus) can be foundin tissue cultures and at least one case where operatorsbecame infected from handlingTrichophyton-infectedtissue cultures has been reported [15]. Such systemshave now been designed and become common prac-tice in many commercial laboratories. The challengefor the next 8 years should be to perfect such qualityassurance systems.

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