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Microbiological examination of six industrial soluble oil emulsion samples
Microbiological exami nation of six ustrial soluble oil emulston samples M. D. Wort, G. I. Lloyd and J. Schofield*
Soluble oil emulsion samples from machine tool sumps were examined for their total bacterial population and for potentially pathogenic enteric and haemolytic bacteria. All the samples contained a large bacterial population in which the most abundant organisms belonged to the genus Pseudomonas, and also a significant number of enteric and haemolytic bacteria. Very low numbers of anaerobic sulphide producing bacteria and no fungi were isolated. The samples were generally in poor condition with free oil and slime clearly visible. In only on instance was the oil/water ratio correct.
Micro-organisms occur in every type of environment which is occupied by man, plants and animals. They are responsible for the breakdown of organic material and this process of mineralization, or the return to the inorganic state of nitrogen, sulphur and carbon from organic molecules, is imperative for the continuation of life on Earth. Soluble oil emulsions, which contain a range of hydrocarbons and other organic molecules such as anti-corrosive agents, are vulnerable to breakdown by micro-organisms and this can produce emulsions which are non-lubricating and corrosive. In contaminated soluble oil emulsions, bacteria are usually the dominant type of micro-organism, but fungi, which generally grow more slowly and prefer a more acid environment than bacteria, may occur if the growth of bacteria is selectively suppressed by anti-bacterial compounds or if the pH falls because of acid production by bacteria.
based primarily on their behaviour on agar containing blood. The most highly pathogenic streptococci, eg Streptococcus viridans, produce a clear zone of haemolysis around the bacterial colony which will have grown on the blood agar. The object of this investigation was to determine the number of bacteria and fungi which were present in the emulsion samples from machine tool sumps and to establish the identity of the predominant organisms. The samples were also examined for the presence of potentially pathogenic streptococci, and for enteric bacteria indicating faecal contamination. Materials and Methods Each sample was taken from the coolant stream on a machine tool in production, except sample 'D' where the machine had been idle for two months.
Bacteria are divided into two great classes, gram positive and gram negative, by a staining procedure devised by Gram. The reaction of the bacteria to the staining proceedure is correlated with the nature of the cell wall. The most important organisms with a strong capability of growing in coolants are gram negative and of these the genus Pseudomonas (Fig 1) plays a major part in emulsion degradation. Members of the enteric group of bacteria (gram negative) such as Escherich& sp, Aerobacter sp, Proteus sp, and Klebsiella sp, are frequently found growing in coolants 1. The presence of Escherichia coli, a normal inhabitant of the intestinal tract of man, may indicate that faecal contamination has taken place. This is important because the related genera Salmonella and Shigella are potentially pathogenic, producing intestinal disease. Enteric bacteria can be selectively isolated on a medium devised by McConkey containing bile salts to which the enteric bacteria are resistant. The gram positive genus Streptococcus (Fig 2) contains pathogenic species which can cause boils, sore throats and dermatitis. The classification of this group of bacteria is * Department of Mechanical, Marine and Production Engineering, Liverpool Polytechnic, Byrom St, Liverpool, UK
Fig I Pseudomona olevorans 525x stereoscan electron mierograph
TRIBOLOGY international February 1976
35
(Table 1). The total number of bacteria in the samples was however less than that reported as normal, where emulsions contain between 15 and 27 × 106 organisms/ml under industrial conditions 1. Further, Duffett et al6, in an examination of 634 samples, noted that counts normally exceeded 106 organisms/ml whilst Weirich ~ observed counts as high as 300 x 106 and 710 x 106 organisms/ml. In the present samples the population size may have been restricted in some cases by the reduction in oil surface area because of emulsion breakdown and separation. Further, compared with the rate of bacterial growth the emulsion samples were old and the bacterial population may have entered a death phase with a concomitant reduction in bacteria brought about by end products of bacterial metabolism, eg organic acids, or by substrate exhaustion eg nitrogen source.
Fig 2 Streptococcus faecalis 22 O00x stereoscan electron micrograph
Bacterial and fungal counts
Total, enteric and haemolytic counts were done on samples, homogenized by shaking, using the Miles and Misra technique 3 using nutrient agar, McConkey agar and blood agar respectively. The principal organisms occurring on the nutrient agar plates were identified to the genus level by reference to Cowan and Steel 3. Anaerobic sulphide producing bacteria were detected semi-quantitatively using the black spot test 4. Fungi were isolated on MYGP agar plates s at pH 4.5 by spreading 0.1 ml of the samples over the agar surface with a glass rod. Results and Discussion
All the samples contained a significant number of bacteria, the total count ranging from 3 x 104 organisms/ml of emulsion in sample C to 3.6 x 106 organisms/ml in sample E
Table 1
The predominant species in all samples belonged to the genus Pseudomonas. This group of bacteria have a strong capability for growing in coolant's. They play a major role in coolant degradation 1 and this is probably because of their enormous biochemical versatility and also the nature of their cell wall. Gram negative cells in general are well adapted for life in aqueous environments because, unlike gram positive bacteria, they retain their degradative enzymes in a highly protective association with the cell wall and because the products of digestion by these enzymes are immediately available to the transport system of the cell, they are not lost into the water 8. /3 haemolytic Streptococci were found in four samples showing that potentially pathogenic bacteria can survice in coolants. Their presence may be of little importance since the presence of streptococci has been considered the result of skin problems rather than the cause of them a. However, airborne streptococcal infections causing septic sore throat and skin infection for example, could occur, because airborne bacterial contamination has been shown to greatly increase in the vicinity of a lathe during its operation 9.
Aerobacter aerogenes was the predominant enteric organism found in samples A - D . Eseherichia eolL an indicator
Description of oil emulsion samples - microbiological properties
Sample
Bacterial population per ml of emulsion
Dominant bacteria
A
7.0 x 104
Pseudomonads
B
1.5 x 106
Pseudomonads
Other bacteria isolated Staphylococci Streptococci Staphylococci Aerobacter
Enteric bacteria per ml of emulsion
/3 Haemolytic bacteria per ml of emulsion
Anaerobic bacteria per ml of emulsion
3.0 x 102 No E.coli 5.5 x 103 No E.coli
1.0x 102
30
3.0 x 101
20
None
20
None
None
3.0 x 104
50
3 . 0 x 104
50
Streptococci Bacilli C
3.0 x 104
Pseudomonads
A ero bac ter
D
7.8 x 105
Pseudomonads
A erobac ter
Pseudomonads
Bacilli Streptococci
E
3.6 x 106
A erobac ter Escherich ia
F
6.3 x 105
Pseudomonads
Streptococci Escherichia Aerobacter
36
TRIBOLOGY international February 1976
2.8 x 102 No E.coli 1.6 x 103 No E.cofi 1.1 x 103 15% E.coli 2.1 x 103 95% E.coli
Table 2
Description of oil emulsion samples - physical properties
Sample Origin
Age of emulsion
Oil/water* ratio pH
Foul odour
Discolouration
Sediment
A B
C a p s t a nlathe 17in Swing Centre lathe Power Saw
4 weeks 5 weeks
1:5 1:3
8.9 8.4
None None
None None
Unknown
1:9
8.6
None
Slight Moderate Blue/Purple None
Special purpose bar turning machine Universal milling machine 9in Swing Centre lathe
14 weeks
1:21
7.5
None
None
None
2 weeks
1:27
7.6
None
None
None
2 years
1:2
8.0
Slight
Heavy Black
Slight
C D
E F
*
None
Free oil
Free water
Slight Moderate Ca 10% v/v Moderate Ca 10% v/v Slight with bacterial slime
None None
Slight with bacterial slime Heavy Ca 50% v/v with bacterial slime
None
None None
Heavy Ca 30% v/v
Measured using a Shell slurrimeter
of faecal contamination, was only isolated from samples E and F where it consisted of 15% and 95% o f the enteric bacteria respectively. No fungi were isolated from any of the samples.
industry then a vast quantity of soluble oil is wasted every day.
The maintenance of a stable oil emulsion is a complex process involving both chemical and physical parameters. Any alteration from the norm can result in instability and separation of oil and water phases. Such a situation can be caused by a heavy bacterial infection as in the six samples examined. Two o f the samples B and F showed marked emulsion phases suggesting total breakdown of the system (Table 2). The main source o f infection is not the water or neat oil of the emulsion but the residual components of the previous charge 4. A new charge of emulsion can pick up a massive infection even after cleaning and draining the system. This has a significant effect on the subsequent life and performance o f the new emulsion. The samples examined in this study were made even more deleterious by the use of incorrect oil:water ratios. Far too rich a mixture appeared to be the general rule 1:30 oil to water ratios are adequate for most engineering applications. If the samples reflect in any way the general practice of UK
1 Bennett, E. O., The deterioration of metal cutting fluids. Progress in Industrial Microbiology. 13(1974) 121 2 Miles, A. A. and Misra, S. S., J. Hygiene, Carnb. 38 (1938) 732 3 Cowan, S. T., and Steel, K. J. Manual for the identification of medical bacteria. Iliffe Books Ltd., London (1967) 206 4 Hill, E. C. In Microbiological aspects of Metallurgy. Ed. by J. D. A, Miller. Medical and Technical Publishing Co Ltd, Aylesbury (1971) 149 5 Wiekerham, L. J. in 'Taxonomy of Yeasts' US Dept. Agric. Tech.
References
Hull. No 1029 1951
6 Duffett, N. D., Gold, S. H., and Weitich, C. L. J. Bacteriology 45 (1943) 3 7
7 Weirich, C. L., Safety Eng. 85 (1943) 36 8 Costerton, J. W., In,ram, J. M., and Cheng, K. J. Structure and Function of the cell envelope of gram-negative bacteria. Bacteriological Reviews 38 (1974) 87
9 Lloyd, G., Lloyd, G. I., and Schofield, J. Enteric bacteria in cutting oil emulsion. Tribology International (February 1975) 27
TRIBOLOGY international February 1976
37
Soluble oil emulsion samples from machine tool sumps were examined for their total bacterial population and for potentially pathogenic enteric and haemolytic bacteria. All the samples contained a large bacterial population in which the most abundant organisms belonged to the genus Pseudomonas, and also a significant number of enteric and haemolytic bacteria. Very low numbers of anaerobic sulphide producing bacteria and no fungi were isolated. The samples were generally in poor condition with free oil and slime clearly visible. In only on instance was the oil/water ratio correct.
Micro-organisms occur in every type of environment which is occupied by man, plants and animals. They are responsible for the breakdown of organic material and this process of mineralization, or the return to the inorganic state of nitrogen, sulphur and carbon from organic molecules, is imperative for the continuation of life on Earth. Soluble oil emulsions, which contain a range of hydrocarbons and other organic molecules such as anti-corrosive agents, are vulnerable to breakdown by micro-organisms and this can produce emulsions which are non-lubricating and corrosive. In contaminated soluble oil emulsions, bacteria are usually the dominant type of micro-organism, but fungi, which generally grow more slowly and prefer a more acid environment than bacteria, may occur if the growth of bacteria is selectively suppressed by anti-bacterial compounds or if the pH falls because of acid production by bacteria.
based primarily on their behaviour on agar containing blood. The most highly pathogenic streptococci, eg Streptococcus viridans, produce a clear zone of haemolysis around the bacterial colony which will have grown on the blood agar. The object of this investigation was to determine the number of bacteria and fungi which were present in the emulsion samples from machine tool sumps and to establish the identity of the predominant organisms. The samples were also examined for the presence of potentially pathogenic streptococci, and for enteric bacteria indicating faecal contamination. Materials and Methods Each sample was taken from the coolant stream on a machine tool in production, except sample 'D' where the machine had been idle for two months.
Bacteria are divided into two great classes, gram positive and gram negative, by a staining procedure devised by Gram. The reaction of the bacteria to the staining proceedure is correlated with the nature of the cell wall. The most important organisms with a strong capability of growing in coolants are gram negative and of these the genus Pseudomonas (Fig 1) plays a major part in emulsion degradation. Members of the enteric group of bacteria (gram negative) such as Escherich& sp, Aerobacter sp, Proteus sp, and Klebsiella sp, are frequently found growing in coolants 1. The presence of Escherichia coli, a normal inhabitant of the intestinal tract of man, may indicate that faecal contamination has taken place. This is important because the related genera Salmonella and Shigella are potentially pathogenic, producing intestinal disease. Enteric bacteria can be selectively isolated on a medium devised by McConkey containing bile salts to which the enteric bacteria are resistant. The gram positive genus Streptococcus (Fig 2) contains pathogenic species which can cause boils, sore throats and dermatitis. The classification of this group of bacteria is * Department of Mechanical, Marine and Production Engineering, Liverpool Polytechnic, Byrom St, Liverpool, UK
Fig I Pseudomona olevorans 525x stereoscan electron mierograph
TRIBOLOGY international February 1976
35
(Table 1). The total number of bacteria in the samples was however less than that reported as normal, where emulsions contain between 15 and 27 × 106 organisms/ml under industrial conditions 1. Further, Duffett et al6, in an examination of 634 samples, noted that counts normally exceeded 106 organisms/ml whilst Weirich ~ observed counts as high as 300 x 106 and 710 x 106 organisms/ml. In the present samples the population size may have been restricted in some cases by the reduction in oil surface area because of emulsion breakdown and separation. Further, compared with the rate of bacterial growth the emulsion samples were old and the bacterial population may have entered a death phase with a concomitant reduction in bacteria brought about by end products of bacterial metabolism, eg organic acids, or by substrate exhaustion eg nitrogen source.
Fig 2 Streptococcus faecalis 22 O00x stereoscan electron micrograph
Bacterial and fungal counts
Total, enteric and haemolytic counts were done on samples, homogenized by shaking, using the Miles and Misra technique 3 using nutrient agar, McConkey agar and blood agar respectively. The principal organisms occurring on the nutrient agar plates were identified to the genus level by reference to Cowan and Steel 3. Anaerobic sulphide producing bacteria were detected semi-quantitatively using the black spot test 4. Fungi were isolated on MYGP agar plates s at pH 4.5 by spreading 0.1 ml of the samples over the agar surface with a glass rod. Results and Discussion
All the samples contained a significant number of bacteria, the total count ranging from 3 x 104 organisms/ml of emulsion in sample C to 3.6 x 106 organisms/ml in sample E
Table 1
The predominant species in all samples belonged to the genus Pseudomonas. This group of bacteria have a strong capability for growing in coolant's. They play a major role in coolant degradation 1 and this is probably because of their enormous biochemical versatility and also the nature of their cell wall. Gram negative cells in general are well adapted for life in aqueous environments because, unlike gram positive bacteria, they retain their degradative enzymes in a highly protective association with the cell wall and because the products of digestion by these enzymes are immediately available to the transport system of the cell, they are not lost into the water 8. /3 haemolytic Streptococci were found in four samples showing that potentially pathogenic bacteria can survice in coolants. Their presence may be of little importance since the presence of streptococci has been considered the result of skin problems rather than the cause of them a. However, airborne streptococcal infections causing septic sore throat and skin infection for example, could occur, because airborne bacterial contamination has been shown to greatly increase in the vicinity of a lathe during its operation 9.
Aerobacter aerogenes was the predominant enteric organism found in samples A - D . Eseherichia eolL an indicator
Description of oil emulsion samples - microbiological properties
Sample
Bacterial population per ml of emulsion
Dominant bacteria
A
7.0 x 104
Pseudomonads
B
1.5 x 106
Pseudomonads
Other bacteria isolated Staphylococci Streptococci Staphylococci Aerobacter
Enteric bacteria per ml of emulsion
/3 Haemolytic bacteria per ml of emulsion
Anaerobic bacteria per ml of emulsion
3.0 x 102 No E.coli 5.5 x 103 No E.coli
1.0x 102
30
3.0 x 101
20
None
20
None
None
3.0 x 104
50
3 . 0 x 104
50
Streptococci Bacilli C
3.0 x 104
Pseudomonads
A ero bac ter
D
7.8 x 105
Pseudomonads
A erobac ter
Pseudomonads
Bacilli Streptococci
E
3.6 x 106
A erobac ter Escherich ia
F
6.3 x 105
Pseudomonads
Streptococci Escherichia Aerobacter
36
TRIBOLOGY international February 1976
2.8 x 102 No E.coli 1.6 x 103 No E.cofi 1.1 x 103 15% E.coli 2.1 x 103 95% E.coli
Table 2
Description of oil emulsion samples - physical properties
Sample Origin
Age of emulsion
Oil/water* ratio pH
Foul odour
Discolouration
Sediment
A B
C a p s t a nlathe 17in Swing Centre lathe Power Saw
4 weeks 5 weeks
1:5 1:3
8.9 8.4
None None
None None
Unknown
1:9
8.6
None
Slight Moderate Blue/Purple None
Special purpose bar turning machine Universal milling machine 9in Swing Centre lathe
14 weeks
1:21
7.5
None
None
None
2 weeks
1:27
7.6
None
None
None
2 years
1:2
8.0
Slight
Heavy Black
Slight
C D
E F
*
None
Free oil
Free water
Slight Moderate Ca 10% v/v Moderate Ca 10% v/v Slight with bacterial slime
None None
Slight with bacterial slime Heavy Ca 50% v/v with bacterial slime
None
None None
Heavy Ca 30% v/v
Measured using a Shell slurrimeter
of faecal contamination, was only isolated from samples E and F where it consisted of 15% and 95% o f the enteric bacteria respectively. No fungi were isolated from any of the samples.
industry then a vast quantity of soluble oil is wasted every day.
The maintenance of a stable oil emulsion is a complex process involving both chemical and physical parameters. Any alteration from the norm can result in instability and separation of oil and water phases. Such a situation can be caused by a heavy bacterial infection as in the six samples examined. Two o f the samples B and F showed marked emulsion phases suggesting total breakdown of the system (Table 2). The main source o f infection is not the water or neat oil of the emulsion but the residual components of the previous charge 4. A new charge of emulsion can pick up a massive infection even after cleaning and draining the system. This has a significant effect on the subsequent life and performance o f the new emulsion. The samples examined in this study were made even more deleterious by the use of incorrect oil:water ratios. Far too rich a mixture appeared to be the general rule 1:30 oil to water ratios are adequate for most engineering applications. If the samples reflect in any way the general practice of UK
1 Bennett, E. O., The deterioration of metal cutting fluids. Progress in Industrial Microbiology. 13(1974) 121 2 Miles, A. A. and Misra, S. S., J. Hygiene, Carnb. 38 (1938) 732 3 Cowan, S. T., and Steel, K. J. Manual for the identification of medical bacteria. Iliffe Books Ltd., London (1967) 206 4 Hill, E. C. In Microbiological aspects of Metallurgy. Ed. by J. D. A, Miller. Medical and Technical Publishing Co Ltd, Aylesbury (1971) 149 5 Wiekerham, L. J. in 'Taxonomy of Yeasts' US Dept. Agric. Tech.
References
Hull. No 1029 1951
6 Duffett, N. D., Gold, S. H., and Weitich, C. L. J. Bacteriology 45 (1943) 3 7
7 Weirich, C. L., Safety Eng. 85 (1943) 36 8 Costerton, J. W., In,ram, J. M., and Cheng, K. J. Structure and Function of the cell envelope of gram-negative bacteria. Bacteriological Reviews 38 (1974) 87
9 Lloyd, G., Lloyd, G. I., and Schofield, J. Enteric bacteria in cutting oil emulsion. Tribology International (February 1975) 27
TRIBOLOGY international February 1976
37