Poor Quality of Oocytes from Xenopus Laevis Used in Laboratory Experiments: Prevention by Use of Antiseptic Surgical Technique and Antibiotic Supplementation

Poor Quality of Oocytes from Xenopus laevis Used in Laboratory Experiments: Prevention by Use of Antiseptic Surgical Technique and Antibiotic Supplementation

Holger-Andreas Elsner,1,2 Hans-Hinrich Hönck,3 Frank Willmann,4 Hans-Jürgen Kreienkamp,3 and Franz Iglauer4

Background and Purpose: Episodic phases of continuous poor-quality oocytes obtained from South American Clawed Frogs (Xenopus laevis) often are observed. In publications dealing with the surgical technique of oocyte removal, the frogs’ robust constitution and resistance against infections provided by magainins are pointed out. For this reason, clean rather than sterile conditions for the surgical procedure are mostly recommended. However, in most instances, antibiotics are added to the buffer medium when in vitro experiments are performed using oocytes. Methods: After a long phase of poor oocyte quality at our facility, involving oocytes that had been obtained by use of a “clean” surgical procedure, we subsequently cultured oocytes in a buffer medium containing the three antibiotics: penicillin G, gentamicin, and streptomycin. Results: During DNA injection experiments, the oocytes developed black spots on their surface by postoperative day two. Pure culture of the gram-negative non-fermentative rod Pseudomonas fluorescens was obtained from the impaired oocytes; the isolate was resistant to the three antibiotics. By contrast, after aseptic surgical removal and culture of oocytes in buffer medium containing the antibiotics tetracycline and gentamicin, perfect oocytes without bacterial contamination were obtained. Conclusion: Whenever impaired oocyte quality is observed, microbial contamination should be considered as a possible cause.

In former times, South African clawed frogs (Xenopus laevis) Despite great experience in rearing X. laevis and in oocyte were mostly used for diagnosis of gravidity in obstetrics, for si- removal from the living animal, we observed a period of poor tus preparations, or for the study of developmental processes in oocyte quality from December 1997 until January 1998. During practices/exercises of physiology. Currently, its most frequent RNA injection experiments, postoperatively healthy-appearing biomedical use is to obtain oocytes for research in molecular bi- oocytes developed marbled spots on the surface (Figure 1). ology, biophysics, and ontogenesis. These changes are reminiscent of the apoptotic stage of normal At the Laboratory Animal Facility, University Hospital Ham- cell aging (4). After only one or two days, the electric resting po- burg, South African clawed frogs have been successfully bred for tential was very low (–10 to –20 mV), wheras healthy oocytes many years. These frogs were maintained in accordance with the (Figure 2) usually remain optically unchanged after one week most recent accomodation recommendations (1), and in compliance and maintain a rather high resisting potential (–30 to –60 mV). with the governmental supervising authority (Hamburg, Ger- In most institutes, researchers complain about the disastrous many), in particular with §11, Tierschutzgesetz. The adult females episodes of continuous (weeks or months) poor oocyte quality had been received from various resources: NASCO (Fort Atkinson, from all donor frogs that belong to the same colony (5, 6). Rear- Wisconsin) or Fa. Dipl. Biol. H. Kähler (Schramsweg 13, 20249 ing conditions, parasitic or other diseases, origin of the frogs, or Hamburg, Germany), or had been bred in our facility (stock: simply the “warm season” are suspected to cause this phenom- Uhh:Xenopus). According to the categories of the National Re- enon (4, 7–10). However, during this period of poor oocyte qual- search Council (2), the hygienic status was “laboratory-reared ity and during former periods, we never found evidence for any standard” or “laboratory-bred standard.” Feeding and husbandry of these suppositions to be causative. Frogs were kept at 20 Ϯ conditions were those described elsewhere (3). Use of these labora- 2ЊC in flow-through or static tanks. As oocytes from different tory animals was indicated and licensed (§6 Abs.1 Satz 2 Nr.4 frogs were involved, genetic reasons, including inbreeding, also TierSchG) by the government office in Hamburg for laboratory were unlikely. At our facility, each X. laevis is used six times at animal experiments (16.07.1996/Lo: G 8151/591-00.33). a maximum. Body weight is monitored before and after surgery. The interval between two surgeries is at least three months. 1Department of Transfusion Medicine, Hannover Medical School, Carl-Neuberg- Frogs were not used if there was any reduction in body weight Str. 1, D-30625 Hannover; 2Institute of Medical Microbiology and Immunology, University Hospital Eppendorf, Martinistr. 52, D-20246 Hamburg; 3Institute prior to the next scheduled surgery. Each frog from our own for Cell Biochemistry and Clinical Neurobiology, University Hospital Eppendorf, stock was at least two years old and weighed Ն 90 g. Those from Martinistr. 52, D-20246 Hamburg; and 4Laboratory Animal Facility, University extramural sources had body weight of at least 120 g. The frogs Hospital Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. Dr. Iglauer’s present address is Tierschutzbeauftragter, Universität Tübingen, had good appetite, activity, and general appearance. There were Calwer Straße 7/4, 72076 Tübingen, Germany. no signs of skin discoloration or other disorders. The frog colony 206 Prevention of Poor Quality of Xenopus Oocytes

Figure 1. Impaired oocytes that were useless for electrophysiologic Figure 2. Oocytes immediately after removal (no signs of impairment). experiments. mens. A short time after surgical removal, all oocytes had been was monitored for skin parasites as described (10). Since 1997, optically unchanged, with distinct black and white hemispheres our own breeding colony has been free of skin nematodes. Those (Figure 2), indicating that impairment was most likely not of extramural sources had been treated by use of methods suit- caused by disease of the donor frog, but developed later. After able for eradication of skin nematodes (10). This was done three defolliculation, marbled spots could be visualized at a seven-fold months before surgery, when the frogs arrived at our facility. magnification after 24 hours (Figure 1). Oocytes that were incu- In publications dealing with surgical oocyte removal tech- bated over 24 hours within the mesovarium remained un- niques, the frogs’ robust constitution has been discussed and changed, suggesting a normal protective effect of this structure. wound infections are rarely described. The resistance against To explain the bacterial growth despite antibiotic supplemen- infection of the surgical wound is mainly prevented by antimi- tation, minimal inhibitory concentrations (MICs) of penicillin G, crobial peptides (magainin) located in the frog’s skin (4, 7, 11). gentamicin, streptomycin, tetracycline, and ciprofloxacin (single For this reason, normal sterile conditions for surgery are re- garded unnecessary, but a “clean surgical technique” is ap- agents) against all Pseudomonas fluorescens isolates were de- praised as adequate (7), which means avoidance of coarse termined on Mueller-Hinton agar (Difco), using E-test strips contamination of the surgical area. However, in most instances, (AB Biodisk, Solna, Sweden). These test strips, which are antibiotics (streptomycin, gentamicin, penicillin G) are added to charged with an increasing concentration of each antibiotic and the buffer medium when in vitro experiments are performed are put on the agar plate, render easy determination of the MIC, which corresponds to the number printed on the strip where the with oocytes (12). At the time of the impaired oocyte quality outline of the growth inhibition zone touches the scale on the event, a “clean surgical technique” was our standard, and the strip. Incubation times were 24 hours at 30ЊC, 24 hours at 35ЊC, combination of streptomycin (100 ␮g/ml), gentamicin (100 ␮g/ and 6 days at 35ЊC. Test were performed at AB Biodisk. Entero- ml) and penicillin G (59 ␮g/ ml) was added to the Barth me- coccus faecalis (ATCC 29212), Staphylococcus aureus (ATCC dium, in which oocytes were kept. 29213), Escherichia coli (ATCC 25922), and P. aeruginosa To prove or exclude microbial contamination of the oocytes, (ATCC 27853) were used for quality control. nine oocyte specimens obtained from four frogs during the pe- Results of antibiotic susceptibility tests are shown in Table 2. riod between December 15, 1997 (approx. two weeks after the After incubation for 24 hours at 35ЊC, there were no visible colo- beginning of the episode) and February 2, 1998 (Table 1) were nies on the agar. However, there was light or very light growth examined microbiologically. The four frogs belonged to a colony after prolonged incubation at 6 days. At these conditions MICs of approximately 40 animals and were randomly chosen. The were lower than those at incubation for 24 hours at 30ЊC, re- oocyte specimens were streaked on 10% sheep blood agar with flecting weaker growth. After incubation at 30ЊC for 24 hours Columbia Blood Agar Base (Difco, Detroit), MacConkey agar and at 35ЊC for 6 days, high MICs that exceeded concentrations (Oxoid, Basingstoke, UK), and for detection of fungi on Kimmig in the Barth medium were found for penicillin G, gentamicin, agar (Merck, Darmstadt, Germany) supplemented with 0.5 % glycerol, 32 ␮g/ml ampicillin (ratiopharm, Ulm, Germany), and 32 and streptomycin. In contrast, low MICs were found for tetracy- ␮g of gentamicin (Refobacin®, Merck, Darmstadt, Germany)/ml. cline and ciprofloxacin. The optimal conditions for susceptibility Plates were incubated at 30ЊC for 48 hours. As indicated (Table testing of P. fluorescens have yet to be determined, because the Њ 1), pure cultures of the non-fermentative gram-negative rod best incubation temperature may be < 30 C. Pseudomonas fluorescens were isolated from all these speci- After repeated isolation of P. fluorescens from impaired oocyte 207 Vol 50, No 2 Comparative Medicine April 2000

Enterobacter

Impaired (24h) Impaired (24h)

NT NT NT Impaired (24h) Impaired (24h) Normal (24–96h) Impaired (24h) NT Impaired (48h)

Normal (24–72h) Normal (48–96h) Impaired (48h) Normal (24–72h) Impaired (72h) Impaired (48h) Normal (48–96h) Normal (48–96h)

NT NT

= in addition,

(i), with spots:

0/20 (24h), 0/10 (48–96h)

2/10 (48h), 0/8 (48–96h) 11/11 (48h)

1/16 (24h), 0/15 (48–96h) 13/13 (48h)

15/15 (48h) 2/14 (24h), 0/12 (48–96h)

1/13 (24h), 0/12 (48–96h)

0/20 (72h)

1/20 (72h)

/frequency

Normal (i), spots (24h) Normal (i–24h)

with spots (72h)

Normal (i–24h),

= injection of cRNA: GIRK;

3 3

(24h) (24h) (i), spots Normal (24h) (24h) (i), spots Normal (24h) (i) Normal (i) NT (48h) (i) spots Normal (48h) (96h) (i) spots Normal (96h) (24h) Spots (24h) Impaired (24h) (48h) (i) spots Normal (48h)

(24h)

(24h) (i), spots Normal (24h) (i) Spots (24h) Impaired (24h) (48h) (i), with Normal spots:

(48h) (i), with Normal spots:

(72h)

(72h) with spots:20/20 (72h)

P. fluorescens P. fluorescens P. P. fluorescens P. P. fluorescens P. P. fluorescens P. P. fluorescens P. fluorescens P. P. fluorescens P.

P. fluorescens P.

P. fluorescens P. fluorescens P. P. fluorescens P.

P. fluorescens P.

A. sobria, Alc. faecalis

= injection of cRNA: SLC1;

,S,G,T NT Normal

Antibiotics Bacteriology Microscopy Electrophysiological

P,S,G P,S,G P,S,G Sterile (24h) Normal (24h) NT

P,S,G P,S,G,T NT Normal

P,S,G (72h) Sterile Normal (i–72h) P,S,G NTNone (48h), spots Normal (72h) NT Normal (i), with spots: P,S,G P,S,G

None

(i) after defollic. 30h after defollic.

30h after defollic.

(48h) after defollic.

Conditions of oocyte removal and results

Table 1. Table

1 2

Artificial contamination

P. fluorescens P. fluorescens P.

P. fluorescens P. fluorescens P. P. fluorescens P.

P. fluorescens P.

Defolliculation

and injection of c-RNA

= skin antisepsis was performed, using povidone iodine, sterile drape, and instruments.

No. timing (timing) (timing) (timing) quality

was isolated in small numbers;

Date Frog Antiseptic

15 Dec 1997 1 - i - P,S,G 26 Jan 1998 2 - i - 02 Feb 1998 3 - i - P,S,G 02 Feb 1998 3 - i - P,S,G 02 Feb 1998 3 - i - P,S,G 02 Feb 1998 323 Feb 1998 - 6 + 48h i - - P,S,G None Sterile (24h) Normal (24h) Normal (24–72h) 09 Feb 1998 423 Feb 1998 + 6 + i i - - P,S,G Sterile (72h) P,S,G Normal (i–72h) Sterile (24h) Normal (24h) Normal (24–96h) 15 Dec 1997 1 -09 Feb 1998 4 24h1998Feb 23 + 6 + - i i - P,S, Skin contact (i) before defollic. 02 Mar 1998 7 + i - P 26 Jan 1998 2 - i - P,S,G 26 Jan 1998 2 -16 Feb 1998 5 i02 Mar 1998 + 7 + i - i

02 Mar 1998 7 +09 Mar 1998 i 8 + - 48h - None NT G,Tspots: with (i), Normal NT Normal (i–72h) Normal (72h) 09 Mar 1998 8 + 24h - G,T NT Normal (i), with spots: 09 Feb 1998 4 +02 Mar 1998 i 702 Mar 1998 + 7 + i i - P,S,G NTspots: with (i), Normal 16 Feb 1998Feb 16 5 + i skin contact (i) before defollic. 02 Mar 1998 702 Mar 1998 + 7 + i i - None NTspots: with (i), Normal

02 Mar 1998 7 + i

09 Mar 1998 8 + 48h - None NT Normal (i–72h) 09 Mar 1998 8 + 24h - None NT Normal (i), with spots:

09 Mar 1998 8 + 72h Skin contact (i) after removal

09 Mar 1998 8 + 24h Skin contact (i) after removal

aerogenes P = Penicillin G; G = gentamicin; S = streptomycin; T = tetracycline; NT = not tested; + = yes; - = no; i = immediately; P 208 Prevention of Poor Quality of Xenopus Oocytes

Table 2. Results of antibiotic susceptibility tests Penicillin G Gentamicin Streptomycin Tetracycline Ciprofloxacin Comment Date of isolation Incubation conditions MIC (g/ml) MIC (g/ml) MIC (g/ml) MIC (g/ml) MIC (g/ml) 12 December 1997 30ЊC, 24h >256 >256 >256 4 0.064 35ЊC, 6d >256 >256 >256 0.75 0.023 lg 26 January 1998 30ЊC, 24h >256 >256 >256 4 0.047 vlg 35ЊC, 6d >256 >256 >256 1 0.008 vlg 2 February 1998 30ЊC, 24h >256 >256 >256 4 0.064 lg 35ЊC, 6d >256 >256 >256 1 0.023 lg 2 February 1998 30ЊC, 24h >256 >256 >256 4 0.064 35ЊC, 6d >256 >256 >256 0.75 0.023 lg 2 February 1998 30ЊC, 24h >256 >256 >256 4 0.064 lg 35ЊC, 6d >256 >256 >256 0.75 0.016 lg 2 February 1998 30ЊC, 24h >256 >256 >256 4 0.064 lg 35ЊC, 6d >256 >256 >256 0.75 0.023 lg 16 February 1998 30ЊC, 24h >256 >256 >256 4 0.064 lg 35ЊC, 6d >256 >256 >256 0.75 0.023 lg 16 February 1998 30ЊC, 24h >256 >256 >256 4 0.064 lg 35ЊC, 6d >256 >256 >256 0.75 0.023 lg 2 March 1998 30ЊC, 24h >256 >256 >256 4 0.064 35ЊC, 6d >256 >256 >256 1 0.023 lg lg = Light growth; vlg = very light growth. cultures, we searched for the source of contamination and iso- Table 3. Skin swab specimens from five healthy frogs from lated this bacterium from skin swab specimens from one of five various containers healthy frogs that had not currently been used as donor frogs, Isolates frequencies related to five frogs are shown in brackets. but were a random sample of the affected colony. Species differ- Non-fermentative gram-negative rods Aeromonas hydrophila (5/5) entiation and isolation frequencies of the other microorganisms A. caviae (3/5) are shown in Table 3. As expected, most isolates were identified Flavobacterium species (2/5) as non-fermentative gram-negative rods, which typically colo- Pseudomonas fluorescens (1/5) Enterobacteriaceae nize aquatic environments. Bacteria were identified by Gram Citrobacter freundii (2/5) staining morphology, and biochemically, using API 20 NE, API Enterobacter agglomerans (2/5) Staphylococci 20 E, or ID 32 STAPH (bioMérieux, Marcy L’étoile, France). S. warneri (3/5) Fungi were identified by micromorphology. Identification as P. Yeasts fluorescens was additionally confirmed by presence of fluores- Candida spp. (1/5) cent pigment at transillumination with UV light after incuba- tio and injected at a concentration of 80 ng of total RNA/ml into Њ tion on Mueller-Hinton agar (Difco) at 30 C for 24 hours. On Xenopus laevis oocytes. Oocytes were kept in Barth medium for frog skin impaired due to parasitism, additional presence of two to four days at 20ЊC. In some instances, antibiotic supple- Pseudomonas sp. and related bacteria often is described (4, 6, mentation was added to the Barth medium (Table 1). For re- 13), whereas on healthy frog skin, the number of colony-forming cordings, oocytes were superperfused with ND-96 medium (96 units may be smaller. Thus, failure to detect P. fluorescens in 4 mM NaCl, 2 mM KCl, 2.5 mM CaCl2, 1 mM MgSO4, 5 mM of 5 specimens may be caused by overgrowth of other microor- HEPES, pH 7.5) and clamped at –80 mV. Measurements of the ganisms and does not rule out its presence. somatostatin agonists on GIRK-currents in high potassium me- Because the presence of P. fluorescens on the frog skin sug- dium were then performed as described (14). gested possible contamination during surgical removal, condi- According to results of the MIC determination, the antibiotic tions were varied and artificial contamination experiments supplement was changed: penicillin G, which has only a narrow were performed. The single steps (including results) are shown antibacterial spectrum and is not active against most gram- in Table 1. For antiseptic removal of oocytes, the ventral area of negative rods, was replaced by the broader-spectrum antibiotic the tricaine-anesthetized (MS-222; 0.15% in water bath) frog tetracycline (50 ␮g/ml). In addition, simultaneous use of both ™ was rubbed with 10% povidone-iodine solution (Betaisodona , aminoglycosides, which have a similar antibacterial spectrum, Lösung, Mundipharma, Limburg, Germany). was reduced to either gentamicin (100 ␮g/ml) or streptomycin After 5 minutes, a sterile drape with a slit and plastic surface (100 ␮g/ml). The new combination of tetracycline and was placed over the frog. Laparotomy was performed, using a aminoglycoside is active against most bacteria, including En- sterile scalpel and forceps through the slit, and wound edges terobacteriaceae, non-fermentative gram-negative rods, and were fixed by clamps to the drape. When the coelom was Mycoplasma spp. Although only tetracycline was active against opened, the ovary flowed onto the drape, and contact with the P. fluorescens, an aminoglycoside was added, as there may be frog’s skin was avoided. After removal of a portion of the ovary, other bacteria that are resistant to tetracycline but not gen- using another set of sterile instruments, the wound was closed tamicin or streptomycin. ® with a double-knotted suture (three separate sutures, PDS II When oocytes were surgically removed for the first time on Feb- 1.5 metric 5/0, Ethicon, Norderstedt, Germany). In addition, ruary 9, 1998 under the improved conditions, using sterile instru- preparation of the oocytes was performed under sterile conditions. ments, and after preoperative skin antisepsis, oocytes remained After surgical removal, oocytes were defolliculated mechani- optically unchanged and were suitable for electrophysiologic ex- cally. The cRNAs coding for the rat somatostatin receptor sub- periments over several days. Although supplementation with peni- types 2 and 3 and the GIRK1 subunit of G-protein coupled cillin G, gentamicin, and streptomycin was not sufficiently active inwardly rectifying potassium channels were mixed in a 1:1 ra- against P. fluorescens, the oocytes remained healthy for a period 209 Vol 50, No 2 Comparative Medicine April 2000

Table 4. Comparison of various antiseptic agents. Agent No. of frogs Microbiology Skin irritation Povidone iodine 5 (25 swabs) 1 swab: P. fluorescens 24 swabs: sterile Not observed Ethacridine lactate 5 (25 swabs) 1 swab: sterile 24 swabs: P. fluorescens Not observed Potassium permanganate 1 (5 swabs) 1 swab: Rhizopus sp.1 4 swabs: sterile Mild Benzalkonium chloride 2 (no swabs taken) Not tested Severe plus 2-propanol 1 Fungus. of five or six days, which emphasizes the importance of skin anti- nations may have synergistic toxicity. Impairment of oocyte sepsis and sterile surgical instruments. In contrast, oocytes that electrophysiologic measurements was not observed with use of were deliberately contaminated either by pure cultures of P. the new antibiotic combination. In addition to appropriate fluorescens or by contact with the frog’s skin, developed the typi- buffer media supplementation, we recommend skin antisepsis cal change in morphology as previously described, regardless of by use of povidone iodine and sterile surgical instruments for whether it was incubated in Barth medium containing the old oocyte removal to reduce the number of microorganisms possi- antibiotic combination (penicillin G, gentamicin, streptomycin) bly resistant to the antimicrobial agents in the buffer. or without antibiotics. At the time before skin antisepsis was introduced, oocytes To find the most appropriate agent for skin antisepsis, anti- were contaminated by P. fluorescens during surgical removal of microbial activity was evaluated during surgery. Pseudomonas oocytes, via contact of the ovary with frog’s skin. Later, in the fluorescens bacteria were suspended in 0.9% NaCl solution Barth medium, P. fluorescens was selected because of its resis- (McFarland standard of 0.5) and were rubbed onto the ventral tance to the previously used antibiotics: penicillin G, gentami- skin of 5 healthy anesthetized female frogs. After 3 minutes, cin, and streptomycin. Since establishment of preoperative skin skin antisepsis was performed as described previously, using antisepsis, use of sterile drape and instruments, and change of 10% povidone iodine solution, 0.1% aqueous solution of the antibiotic supplement in the Barth medium (either tetracy- ethacridine lactate (Entozon® N, Hoechst, Frankfurt, Germany), cline [50 ␮g/ml] plus gentamicin [100 ␮g/ml] or tetracycline [50 0.1 % aqueous solution of potassium permanganate, or a com- ␮g/ml] plus streptomycin [100 ␮g/ml]), development of marbled mercially available disinfectant containing 0.025% benzalko- spots and premature collapse of electric resting potential was nium chloride plus 63% 2-propanol (Cutasept F®, Bode, no longer observed in oocytes. Hamburg, Germany). After 5 minutes, 5 swab specimens of the In conclusion, we recommend these measures to prevent poor treated skin area of each frog were taken and streaked on 10% quality of Xenopus oocytes in laboratory experiments. Whenever sheep blood agar and nutrient agar (Standard I-Nähragar, impaired oocyte quality is observed, microbial contamination Merck, Darmstadt, Germany) supplemented with 3% saponin should be considered as a possible reason. (Fluka, Buchs, Switzerland), 3% Tween 80 (Merck), 0.1% L-his- tidine, and 0.1% L-cysteine. In addition, swab specimens were washed in 10 ml of nutrient broth (Standard I-Nährbouillon, Acknowledgment Merck) with identical supplements as those used for the nutri- The support by Åsa Karlsson and Anne Bolström, AB Biodisk, Swe- ent agar. Incubation time was 7 days at 20 to 22ЊC. As indicated den, who performed antibiotic susceptibility testing of Pseudomonas (Table 4), povidone iodine solution was the only agent that was fluorescens isolates at various incubation conditions, using the E-test, is gratefully acknowledged. effective and well tolerated by the frog’s skin, whereas the other agents were either provided insufficient antisepsis or were harmful to the frogs. References As indicated (Table 1), correlation of bacterial isolation and 1. Scharmann, W. (Moderator), F. Gruber, and F. Iglauer. 1993. impaired oocyte quality suggest the causative role of P. fluorescens. South African clawed frogs (Xenopus laevis), p. 65–70. In P. N. Pure cultures of P. fluorescens were isolated from impaired oocytes O’Donoghue (ed.), The accommodation of laboratory animals in that were obtained before establishment of sterile surgical re- accordance with animal welfare requirements. Proceedings of the moval, and were re-isolated from artificially contaminated cul- International Workshop held at the Bundesgesundheitsamt, Ber- lin 17–19 May 1993. tures, thus proving the causative role of this bacterium via 2. National Research Council. 1974. Amphibians: Guidelines for fulfilment of the Henle-Koch postulates. Yet, in future oocyte the breeding, care, and management of laboratory animals. A re- experiments, it must be considered that other gram-negative port of the subcommittee on amphibian standards. Printing and rods also may affect oocytes; impaired oocyte quality was asso- Publishing Office, National Academy of Sciences, Washington, D.C. 3. Hilken, G., J. Dimigen, and F. Iglauer. 1995. Growth of Xeno- ciated with isolation of Aeromonas sobria and Alcaligenes pus laevis under different laboratory rearing conditions. Lab. Ani- faecalis from two specimens dated March 9, 1998 that had been mal. 29:152–162. deliberately contaminated with frog’s skin in Barth medium 4. Richter, H. P. 1997. Verwendung von Xenopus-Eizellen, p.53– without antibiotics. 98. In Society for Laboratory Animal Science, G. Hilken, F. Iglauer, Although tetracycline plus an aminoglycoside cover a broader and H.P. Richter (eds.), Der Krallenfrosch Xenopus laevis als Labortier: Biologie, Haltung, Zucht und experimentelle Nutzung. spectrum of antibacterial activity, in some instances, growth of mi- Ferdinand Enke Verlag, Stuttgart, Germany. croorganisms may also occur in association with this combination, 5. Riedesel, H. 1998. Personal communication. requiring use of other broad-spectrum antibiotics (e.g., 6. Nicklas, W. 1998. Personal communication. ciprofloxacin, chloramphenicol, and rifampin) or antifungal 7. Goldin, A. L. 1992. Maintenance of Xenopus laevis and oocyte injection. Methods Enzymol. 207:266–279. agents. For cell cultivation experiments, it has to be considered 8. Brayton, C. 1992. Wasting disease associated with cutaneous that high concentrations of antibiotics and antifungal agents and renal nematodes, in commercially obtained Xenopus laevis. are potentially harmful to eukaryotic cells and that their combi- Ann. NY Acad. Sci. 653:197–201. 210 Prevention of Poor Quality of Xenopus Oocytes

9. Iglauer, F., and G. Hilken. 1997. Krankheiten und Narkose, 12. Laskey, R. A. 1970. The use of antibiotics in the preparation of p.39–46. In Soceity for Laboratory Animal Science, G. Hilken, F. amphibian cell cultures from highly contaminated material. Iglauer, and H.P. Richter (ed.), Der Krallenfrosch Xenopus laevis J. Cell Sci. 7:653–659. als Labortier: Biologie, Haltung, Zucht und experimentelle 13. Cunningham A. A., A. W. Sainsbury, and J. E. Cooper. 1996. Nutzung. Ferdinand Enke Verlag, Stuttgart, Germany. Diagnosis and treatment of a parasitic dermatitis in a laboratory 10. Iglauer, F., F. Willmann, G. Hilken, et al. 1997. Anthelminthic colony of African clawed frogs (Xenopus laevis). Vet. Rec. 29: treatment to eradicate cutaneous capillariasis in a colony of South 640–642. African Clawed Frogs (Xenopus laevis). Lab. Anim. Sci. 47: 14. Kreienkamp, H. J., H. H. Hönck, and D. Richter. 1997. Cou- 477–482. pling of rat somatostatin receptor subtypes to a G-protein gated 11. Levison, M. E., P. G. Pitsakis, P. L. May, et al. 1993. The bac- inwardly rectifying potassium channel (GIRK1). FEBS Lett. tericidal activity of magainins against Pseudomonas aeruginosa 419:92–94. and Enterococcus faecium. J. Antimicrob. Chemother. 32:577–585.

211