pad culture studies on skin surfaces

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HOLT, R. J. (1966). J . uppl. Bact. 29 (3). 626-630. Pad Culture Studies on Skin Surfaces R. J. HOLT Queen May's Hospital for Children, Carshalton, Surrey, England (Received 6 July, 1966) SUMMARY. A method is described for obtaining replicate velvet pad cultures of skin and other surfaces using malleable pads. This enables the bacterial flora of very irregular surfaces to be charted, and multiple replications on a range of selective media to be made. The application of the method to a number of clinical problems is reported. FOR MANY YEARS it has been recognized that the skin surface is permanently colonized by bacteria ; little has been reported on the species present or their distribution and location, although it is well known that their numbers may be large in certain sites, notably the axillary, inguinal and perianal recesses. This lack of precise information became apparent while investigating, in 1959, the properties of several skin disinfecting compounds which had been formulated to assist in the control of ward and theatre cross infection. It is for this reason that an attempt has been made to evolve a method for sampling skin surfaces which would not only provide information on the number and species of organisms present but their exact location on the skin surface. The most obvious method used by many previous workers-rubbing woollen swabs moistened with saline or broth over a demarcated area of skin-possesses so many disadvantages that it need not be considered further. Others, introduced for the sampling of floors and hard surfaces, such as those of Williams (1949) or Foster (1960), provide more information but are difficult to use when curved or indented surfaces have to be sampled. The same applies to the fabric pad technique reported by Rubbo & Dixson (1960) for sampling fabrics, or that of Gentles (1956) for the isolation of dermato- phytes from floors. The sticky-backed cellophane tape method introduced by Thomas (1961) especially for the sampling of skin surfaces suffers from the disadvantage that the tape is not easy to handle and there is danger that the latex compound may affect some species of organisms. Fluffy blotting paper gives good replication of surface flora, but has disadvantages in use; when moistened with broth it tears readily, particularly if moulded round acute curves. Nevertheless, since it is cheap and therefore need not be resterilized, it is useful for pad culturing relatively flat surfaces. There is no doubt, however, that velvet pads as used by Lederberg & Lederberg (1952) for replication of agar cultures possess the great advantage that a sufficient number of organisms attach themselves to the velvet to enable culture plates to be successively implanted. If, therefore, selective media are employed a great deal more information about the skin flora can be obtained than by any other method. But the hard curved applicator is not suitable for most areas of the human skin;

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Page 1: Pad Culture Studies on Skin Surfaces

HOLT, R. J. (1966). J . uppl. Bact. 29 (3). 626-630.

Pad Culture Studies on Skin Surfaces

R. J. HOLT Queen May ' s Hospital for Children, Carshalton, Surrey, England

(Received 6 July, 1966)

SUMMARY. A method is described for obtaining replicate velvet pad cultures of skin and other surfaces using malleable pads. This enables the bacterial flora of very irregular surfaces to be charted, and multiple replications on a range of selective media to be made. The application of the method to a number of clinical problems is reported.

FOR MANY YEARS it has been recognized that the skin surface is permanently colonized by bacteria ; little has been reported on the species present or their distribution and location, although it is well known that their numbers may be large in certain sites, notably the axillary, inguinal and perianal recesses. This lack of precise information became apparent while investigating, in 1959, the properties of several skin disinfecting compounds which had been formulated to assist in the control of ward and theatre cross infection.

It is for this reason that an attempt has been made to evolve a method for sampling skin surfaces which would not only provide information on the number and species of organisms present but their exact location on the skin surface. The most obvious method used by many previous workers-rubbing woollen swabs moistened with saline or broth over a demarcated area of skin-possesses so many disadvantages that it need not be considered further. Others, introduced for the sampling of floors and hard surfaces, such as those of Williams (1949) or Foster (1960), provide more information but are difficult to use when curved or indented surfaces have to be sampled. The same applies to the fabric pad technique reported by Rubbo & Dixson (1960) for sampling fabrics, or that of Gentles (1956) for the isolation of dermato- phytes from floors. The sticky-backed cellophane tape method introduced by Thomas (1961) especially for the sampling of skin surfaces suffers from the disadvantage that the tape is not easy to handle and there is danger that the latex compound may affect some species of organisms. Fluffy blotting paper gives good replication of surface flora, but has disadvantages in use; when moistened with broth it tears readily, particularly if moulded round acute curves. Nevertheless, since it is cheap and therefore need not be resterilized, it is useful for pad culturing relatively flat surfaces.

There is no doubt, however, that velvet pads as used by Lederberg & Lederberg (1952) for replication of agar cultures possess the great advantage that a sufficient number of organisms attach themselves to the velvet to enable culture plates to be successively implanted. If, therefore, selective media are employed a great deal more information about the skin flora can be obtained than by any other method. But the hard curved applicator is not suitable for most areas of the human skin;

Page 2: Pad Culture Studies on Skin Surfaces

626 R. J. Holt

something that can be moulded to it is required. For this reason the suggestion by my colleague, Mr. B. A. Saggers, that the velvet be stuck on heavy foil was adopted.

The making of such pads and their use for sampling various areas of skin form the subject of this paper.

Materials and Methods (a) Preparation of pads

Trials with a wide range of fabrics indicated that good quality velvet velour was the most satisfactory. It should have a deep, close-knit pile; pale yellow or beige velvet was selected, since on boiling prior to sterilization less dye was leached out than from the more brightly coloured fabrics. Because many of the dyes used in the textile trade have antibacterial or antifungal properties, new batches of velour must be tested to ensure that the particular dye used has no such activity.

The velvet is cut to size and shape dictated by the area of skin to be cultured and the area of culture medium available. It is then glued firmly to heavy grade aluminium foil with a contact adhesive. UHU (UHU-Werk, H.V.M. Fischer, BUhl/ Baden, W. Germany) was found to stand up best to the repeated sterilization suggested below. The foil is then trimmed to the velvet outline, with a flap left to facilitate handling. The foil backing may be marked with spirit ink to indicate orientation of the skin cultures. The pad is sealed in a paper sterilization beg (D.H.P. No. 5 Sterilization Bag, with heat-seal closure; E. 5. & A. Robinson Ltd., Bristol 1) and the pack sterilized by hot air at 160" for 1 h.

(b) Use of IMd Immediately before use the bag is opened by slitting one end, the pad removed with aseptic precautions and laid, velvet upwards, in a sterile dish. Just sufficient sterile broth is pipetted evenly on the pad to moisten the surface; the exact amount absorbed will vary slightly with different batches of velvet; 1 m1/20 om2 is usually adequate. The pad can now be returned to the opened bag and taken to the patient. After inspecting the site under test, the moistened pad is applied firmly and evenly, left in contact for a few seconds and removed by peeling off; it is then carefully restored to the bag. Care must be taken not to slide the pad across the skin surface, otherwise the eventual colonial growth may show smears or 'tailing' of growth. As soon as practicable the pad is inoculated on to a suitable nutrient agar plate; here again, the pad should be applied and removed evenly, without lateral movement; it should be pressed gently on to the agar. Excess pressure will rupture the agar surface and may leave blank areas on the final culture map, since a fYm of agar will adhere to the pad.

(c) Washing and sterilization of pad After use the pad is placed in an open bowl of water and autoclaved a t 121" for 20 min. When cool it is smoothed out and dried over gentle heat. The velvet pile may become flattened during these procedures but can be raised again by gentle combing with a wooden spatula. The pad is now ready for bag sealing end resteriliza tion.

Page 3: Pad Culture Studies on Skin Surfaces

Pad cultures from skin surfaces 627

(d) Media A range of culture dishes may be used, according to the area of surface under examination. Conventional 4 in. Petri dishes, the larger 6 in. dishes and 12 in2 plates (the type used for multiple antibiotic assays) have served in this department. The 6 in. dish is probably suitable for most investigations; the large square plate is more difficult to keep sterile and the wide agar area, although advantageous for large pads, is liable to split or disrupt under relatively gentle pressure.

Any medium that can be stiffened with agar can be adapted to this technique; the one essential is that sufficient agar (at least 2% of New Zealand or its equivalent) is used to form a stiff gel; this greatly facilitates the even application of the velvet pad. The following media have been used a t various times: (1) ordinary nutrient agar for general purposes; the proportion of agar may be raised to 4% or 6% where actively swarming Proteus spp. are likely; (2) MacConkey or desoxycholate-citrate agar; (3) 10% sterile milk agar; (4) phenolphthalein diphosphate agar; ( 5 ) nutrient agar with various antibiotics added, in the search for drug resistant strains; (6) agar with 8% of added salt; (7) gentian violet horse blood agar; (8) tomato-juioe agar (Kulp’s medium) ; (9) Czapek-Dox agar, with 500 1J.g of each of penicillin and strepto- mycin/ml added.

Results The technique can be adapted to meet the requirement of almost any studies on surface colonization due to normal commensal flora, infecting micro-organism or local contaminants. It would appear that the colonies which grow on agar plates after pad impression originate from small groups or microcolonies on the skin surface. This explains why pad impressions may be made from the same pad on a series of media plates. As many as 4 successive cultures can be prepared in this way; if a range of differential media is used, complementary maps of the same area may be prepared, showing the distribution of different bacterial genera. Delineation of growth areas of bacteria with widely varying cultural characteristics is otherwise not always possible. Examples of the application of the technique are given below.

(a) Bacteria on the skin round surgical lesions Pad cultures were used to locate the colonies of saprophytic and pathogenic bacteria such as staphylococci, enterococci, coli-aerogenes bacteria and pseudomonads in relation to unhealed surgical lesions.

An experiment of this nature is illustrated in Plate 1, which shows the skin flora in the region of an infected abdominal wound 3 days after operation. It had been covered with a dry sterile gauze dressing under pressure taping. The wound edges were heavily colonized with penicillin resistant Staphylococcus aureus ; coli-aerogenes bacteria and Staph. albus were also present.

(b) Survival of bacteria on the skin Overnight broth cultures of Staph. aureus and Escherichia coli Type I were diluted in broth to a h a 1 concentration of c . 1 x 108 cells/ml. Of these, 0.1 ml was applied with a throat swab in a broad band to a marked area of the flexor surface

Page 4: Pad Culture Studies on Skin Surfaces

R. J. Holt

of the forearms of 3 volunteers (two adult males, one adult female) and allowed to dry. Pad replicates of these areas taken after 5 min and then a t hourly intervals and cultured on nutrient agar gave colony counts after 24 h at 37" indicated in Table 1. The area had been protected between pad applications by a sterile gauze dressing.

TABLE 1 The survival of Staphylococcus aureus and Escherichia coli

on the skin

Staph. aureua colony countlstreak E . coli colony count/streak from subject from subject

M.P. R.H. B.S. M.P. R.H. B.S.

Period (h) after

inoculation I *

> I A >

0 96 410 244 110 236 160 1 2 3 4 6 8

10

46 266 170 1 7 3 12 160 21 0 1 0 16 24 12 0 1 0 6 74 2 0 0 0 0 9 1 0 0 0

0 0 0 0

The rapid disappearance of E. coli from these surfaces confirmed the pioneer experiment of Colebrook (1941) ; it contrasted with the more prolonged survival of Staph. aurew.

To eliminate possible fallacies in the experiment, three further tests were performed.

(i) Similar streaks of both organisms were made on forearm skin and on glass plates with slightly irregular surfaces (non-reflecting picture frame glass). Pad cultures were made at hourly intervals. The skin inocula disappeared from cultures at similar times to the earlier experiment, but both Staph. aurew and E . coli persisted, although in reduced numbers, for 10 h on the glass surface.

(ii) It was necessary to ensure that repeated pad cultures on the same area of inoculum did not mechanically diminish the colony count. Long streaks (10 om) of Staph. aureus and E . coli were made on a forearm; narrow pads (c. 1.5 cm wide) were applied at hourly intervals so that successive areas were sampled along the original streak. Results similar to the earlier experiments were obtained. The E. coli showed marked reduction within 2 h and disappeared soon after; the Staph. aureus cultures persisted considerably longer.

(iii) The first experiment was repeated with 4 culture streaks on a forearm. Two strains of Staph. aurew were used, one alien to the volunteer (R.H.) and one an autogenous strain from his nose. One E. coli strain was alien and the other a faecal strain isolated from his faeces. No difference was noted in the rate of diminution of colony counts. Both strains of E. coli disappeared rapidly, and both strains of Staph. aurew persisted considerably longer.

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Pad cultures from skin surfaces 629

During examination of the pad cultures from these 3 volunteers it was noted that the two adult males (R.H. and B.S.) had very low colony counts of normal skin flora, mainly Staph. albus. M.P., an adult female, had a high background count, again largely Staph. albus. Further pad cultures were taken from similar areas (each 75 cm2) of the left forearms of 5 more volunteers, three adult female and two adult males, with the results shown in Table 2. It is difficult to account for the wide

TABLE 2

of normal subjects Typical colony counts from pad cultures of the skins

Subject Colony count of normal flora/cmz of skin

M.P.

1M.R. R.H. [g t .

11.4 0.73 0.015 0.03 0.2 0.19 1.5

~ R . M . 0.012

variation in colony count between these healthy adults. M.C., a male, had an arm noticeably more hairy than that of the other males; M.P., a female, was wearing a jumper of synthetic fibres ; the other 3 females had bare arms beneath starched white coats.

Discussion The malleable pad culture technique offers a simple means of making colony counts of pathogenic and commensal flora on the body surface. The various types of bacteria can be accurately located, and this is of particular value when investigating the proximity of natural orifices or surgical wounds and traumatic lesions. Differential culture media can be employed, thus allowing each genus to be identified and counted individually and enabling clear track to be kept of the altering relationships between each area of colonization. Pad replicates on non-selective media, before and after serial culture on selective media, demonstrated that no undue proportion of bacteria was lost, and that inhibitory agents were not carried over to succeeding culture plates.

The surprisingly rapid disappearance of coli-aerogenes bacteria from skin surfaces, as reported by Colebrook (1941), was confirmed, and the persistent survival of Staph. aurew in similar sites was demonstrated. The mechanics of this elimination are not clear, but i t is noteworthy that the same results are found with both auto- genous and heterogenous strains of E . coli and of staphylococci.

In addition to the experiments outlined above, a number of other uses of the technique have been made. The flora of hard surfaces, such as the inside of infant incubators, have been extensively studied by this method and the eficiency of ward sterilization procedures checked. Colony maps of skin of other areas of the

Page 6: Pad Culture Studies on Skin Surfaces

630 R. J. Holt

body, such as the face, and integrated photographs showing the distribution of bacteria and tracing by these organisms the spread of nasal and salivary secretions, have been prepared. The padding technique has also been used with considerable effect during an investigation into the properties of an iodophore skin disinfectant (Saggers & Stewart, 1964).

It is a pleasure to acknowledge the invaluable advice of Professors G. T. Stewart and R. A. Hare. The author is grateful for many fruitful discussions with Mr. B. A. Saggers, for the willing co-operation of ward staff, especially Sisters D. Colbourne, B. Mitchell and I. B. Stones, and for the enthusiastic technical assistance of Mr. Robin Richardson.

References COLEBROOK, L. (1941). The disinfection of skin. Bull. War Med. 11, 2, 73. FOSTER, W. D. (1960). Environmental staphylococcal contamination. Lancet 1, 670. GENTLES, J. C. (1956). The isolation of dermatophytes from floors of communal bathing places.

LEDERBERQ, J. & LEDERBERQ, E. M. (1952). Replica plating and indirect selection of bacterial

RUBBO, S. D. & DIXSON, S. (1960). A contact-plate technique for determining bacterial

SAQQERS, B. A. t STEWART, G. T. (1964). Polyvinyl-pyrrolidone-iodine: an msessment of anti-

THOMAS, M. (1961). The sticky film method of detecting skin staphylococci. Mon. Bull. Miniat.

WILLIAMS, R. E. 0. (1949). Examination of floor dust for haemolytic streptococci. J. Hyg..

J . clin. Path. 9, 374.

mutants. J. Bact. 63, 399.

contamination of fabrics. Lancet ii, 394.

bacterial activity. J. Hyg., Camb. 62, 509.

Hlth 20, 37.

Camb. 47, 416.

Explanation of Plate PLATE 1. Colonies from velvet pad cultures from the skin near an infected abdominal wound

( a ) on plain nutrient agar (mainly coli-aerogenes bacteria and staphylococci); (b) on gentian violet agar (coli-aerogenes bacteria only); (c) on nutrient agar containing 10 pg of benzyl penicillin/ml (coli-aerogenes bacteria and penicillin resistant staphylococci). ( x 0.75).

Page 7: Pad Culture Studies on Skin Surfaces

Journal of Applied Bacteriology, Vol. 29, Part 3

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R . J. HOLT - PAD CULTURES FROM SKIN SURFACES. PLATE 1

Bact fp 630