COMPARATIVE BACTERIOLOGICAL STUDIES
By Prof. F. Marzullo - Division of Clinical Research - Istituti Ospedalieri - Modena

Studies have been carried out on behalf of CISP the Italian Vitreous Enamel Association on the bacteriological properties of surfaces of various materials, both native and specially treated, and stored in a refrigerator. The studies were intended to show

1) The level of spontaneous infection in contact with milk, broth or meat;
2) The level of infection provoked by contact with known bacterial cultures;
3) The ease of decontamination by physico-chemical agents

The following materials were examined:

1. Stainless Steel
2. Anodized aluminium
3. Native aluminium
4. Painted aluminium
5. Enamelled sheet iron
6. Painted sheet iron
7. ABS Polymer
8. Polystyrol
9. PTFE

Materials and Methods
Nine types of plates were used (50x50x12.5 mm).
They were sterilized by UV-irradiation and placed in Petri dishes which had been sterilized by heating.

A) Spontaneous infection
This was followed by studying the development of the spontaneous bacterial flora present on meat, milk and broth. The bacterial load of the three materials was determined initially and after 24-48-72 and 144 hr.

B) Provoked infection
was followed by studying the development of colonies of known titre (i.e. number of organisms) of Staphylococcus pyogenes, Escherichia coli and Proteus mirabilis for 24, 48, 72 and 144 hr.

C) Ease of decontamination
This was studied by following bacterial growth (at 24, 48, 72 and 144 hr.) on surfaces treated with soap and water; water and acetic acid; water and bicarbonate, applied with a brush or cloth.
A cloth or soft brush was preferred so as to avoid scratches which would prevent thorough cleaning and so favour the retention of bacteria.
This phenomenon was separately studied and we showed that even minimal abrasions led to this, particularly with surfaces of ABS, painted iron, polystyrol, painted aluminium and PTFE.
There was little effect with stainless steel or anodized aluminium and none on enamelled iron. In order to give a better picture of any changes, the foodstuffs tested (milk, broth and meat) were first held in an incubator at 37°C until a sufficiently high bacterial level was reached (900 x 107 c.c.). Fifteen tests were carried out for each investigation.

1st EXPERIMENT - SPONTANEOUS INFECTION
Virtually identical results were obtained with milk, broth and meat and they are therefore unified. Table 1 gives the mean bacterial levels found in the 15 tests.

TABLE 1
SURFACE
Bacterial level (x 10-3)
0 ore
24 ore
48 ore
72 ore
144 ore
Stainless steel
5.030
3.050
3.090
3.030
3.020
Anodized aluminium
5.070
3.900
4.010
3.900
4.000
Native aluminium
5.070
3.090
3.090
4.050
3.000
Painted aluminium
5.030
4.000
3.070
3.050
2.900
Enamelled steel
5.070
2.015
2.000
2.020
2.020
Painted iron
5.070
3.100
3.060
3.060
3.060
ABS polymer
5.070
3.025
3.070
3.090
3.025
Polystyrol
5.070
4.000
4.000
4.070
3.090
PTFE
5.080
3.000
3.000
3.000
3.000
N.B.: The figures are in thousands

2nd EXPERIMENT - PROVOKED INFECTION
Here too the results were virtually constant and those from the 15 tests are averaged in Tables 2, 3 and 4.

TABLE 2
SURFACE
STAPHYLOCOCCUS PYOGENES - 10-3 x no. cells at
0 ore
24 ore
48 ore
72 ore
144 ore
Stainless steel
200.000
50.000
40.000
30.000
20.000
Anodized aluminium
200.000
50.000
50.000
40.000
20.000
Native aluminium
200.000
45.000
45.000
40.000
35.000
Painted aluminium
200.000
30.000
15.000
15.000
7.000
Enamelled steel
200.000
15.000
5.000
1.000
650
Painted iron
200.000
20.000
20.000
10.000
10.000
ABS polymer
200.000
30.000
28.000
20.000
10.000
Polystyrol
200.000
20.000
15.000
15.000
7.500
PTFE
200.000
60.000
60.000
60.000
50.000
N.B.: The figures are in thousands

TABLE 3
SURFACE
ESCHERICHIA COLI - 10-3 x no. cells at
0 ore
24 ore
48 ore
72 ore
144 ore
Stainless steel
300.000
75.000
50.000
30.000
25.000
Anodized aluminium
300.000
68.000
60.000
18.000
18.000
Native aluminium
300.000
70.000
60.000
50.000
50.000
Painted aluminium
300.000
40.000
20.000
16.000
9.000
Enamelled steel
300.000
18.000
10.000
5.000
5.000
Painted iron
300.000
40.000
40.000
10.000
10.000
ABS polymer
300.000
30.000
30.000
30.000
18.000
Polystyrol
300.000
26.000
14.000
14.000
10.000
PTFE
300.000
75.000
50.000
50.000
50.000
N.B.: The figures are in thousands

TABLE 4
SURFACE
PROTEUS MIRABILIS - 10-3 x no. cells at
0 ore
24 ore
48 ore
72 ore
144 ore
Stainless steel
400.000
100.000
90.000
60.000
50.000
Anodized aluminium
400.000
90.000
70.000
70.000
70.000
Native aluminium
400.000
90.000
60.000
60.000
50.000
Painted aluminium
400.000
50.000
30.000
15.000
7.000
Enamelled steel
400.000
28.000
14.000
10.000
850
Painted iron
400.000
60.000
40.000
30.000
10.000
ABS polymer
400.000
70.000
60.000
60.000
60.000
Polystyrol
400.000
70.000
50.000
50.000
15.000
PTFE
400.000
90.000
90.000
70.000
50.000
N.B.: The figures are in thousand

3rd EXPERIMENT - DECONTAMINATION
Here too the results were virtually constant whichever detergent was used (soap and water; water and acetic acid; water and bicarbonate). The results of the 15 tests under each condition are averaged in table 5.

TABLE 5
SURFACE
DECONTAMINATION - 10-3 x no. cells at
0 ore
24 ore
48 ore
72 ore
144 ore
Stainless steel
900.000
5.000
5.000
5.000
4.000
Anodized aluminium
900.000
8.000
7.000
7.000
6.000
Native aluminium
900.000
9.000
9.000
9.000
9.000
Painted aluminium
900.000
5.000
3.000
3.000
3.000
Enamelled steel
900.000
1.000
700
600
600
Painted iron
900.000
7.000
5.000
5.000
5.000
ABS polymer
900.000
5.000
5.000
4.000
3.000
Polystyrol
900.000
4.000
4.000
4.000
4.000
PTFE
900.000
7.000
7.000
3.000
3.000
N.B.: The figures are in thousand

Comment
The following conclusions can be drawn from these results:

A) Spontaneus infection
On all the surfaces the bacterial level undergoes a fall which is apparent even at 24 hr. The greatest fall, which reached or exceeded 50% of the initial value, was on enamelled steel.

B) Provoked infection
No matter which organism was studied there was a rapid and significant inhibition of microbial growth. The greatest effect was on enamelled steel.

C) Decontamination
There was good decontamination at every surface no matter how infected or washed. Again this was most marked with enamelled steel.
It must be pointed out that the action of cold alone is sufficient to inhibit bacterial life independently of the material studied. However the constancy with which some surfaces show different degrees of bacterial inhibition compared with others leads to a consideration of a direct effect. It may readily be imagined that greater smoothness, greater compactness, a greater or less possibility of roughening or still worse of abrasions plays a role of first importance in decontamination. As far as spontaneous or provoked infection is concerned however it is necessary to invoke some chemical or physical factor which acts either directly on bacterial metabolism or by producing changes in the medium used (i.e. in the food or culture medium) and so lead to an inhibition of bacterial life.
Similar findings were made in a study of the mould flora which is often present in refrigerators and which is so difficult to eliminate once it becomes established. The degrees of inhibition which were found with bacterial cultures were closely followed with the moulds i.e.

1) Enamalled steel maximum inhibition
2) Painted iron medium inhibition
3) Painted aluminium medium inhibition
4) ABS polymer no inhibition
5) Polystyrol no inhibition
6) Native aluminium no inhibition
7) Stainless steel no inhibition
8) Anodized aluminium no inhibition
9) PTFE no inhibition

The most significant part of this work is probably represented by these data taken together with those in the experimental part.

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