Determination of microorganisms in coolants by the use of dip-slides

Schülke & Mayr
Determination of microorganisms in coolants by the use of dip-slides
1.1 Introduction
A long standing time of coolants can only be achieved in practice if care measures, such as e.g. the
addition of biocides, are carried out in good time. The problem with this is finding the right time for the
addition. This can be done a) by means of regularly checking the level of the biocidal substances, and b)
by checking the microbe counts. What is ideal is a combination of the two.
In practice, however, regular checking of the biocide level is used only for monitoring large central plantswith volumes over 50 m3, as the analysis involved is very complicated and costly (50-150 €/analysis,depending on substance). With weekly checks, analysis costs that exceed the coolant costs for anindividually filled machine can therefore mount up very quickly.
For individually filled units it is more sensible to keep a coolant check log in which the most important,simply measured, evaluation criteria such as changes in smell, appearance, pH, concentration, corrosionand microbe count, and the measures taken, are documented weekly. By taking these factors together, amicrobial problem can be recognised at an early stage and the necessary counter-measures can be takenin good time.
The simplest, most efficient and cost-effective method at present for determining the microbe count is theuse of so-called dipslides. With these, the microorganisms can be subdivided selectively into bacteria,yeasts and fungi, which is crucial for the selection of the required biocide. Dipslides are generally plastic nutrient medium carriers, coated on one side with a TTC agar (growth ofbacteria, see Table 1) and on the other side with a Rose Bengal agar (growth of yeasts and moulds,Table 1).
Rose Bengal agar
Table 1: Composition of a dipslide
Since excessive bacterial colonisation can adversely affect the growth of fungi and yeasts, antibiotics areoften added to the Rose Bengal agar (in the example chloramphenicol and gentamicin). However, thisdoes not affect the growth of fungi in any way.
The agar surfaces contain no toxic components that could be transferred to the products being tested.
In addition, the dipslides are supplied with a transparent and unbreakable protective tube. Subsequentcontamination is therefore excluded. The result can be evaluated without the tube being opened. A risk ofcontamination for the employees is thus avoided. Schülke & Mayr
1.2 Use of dipslides
A great advantage of dipslides is their ease of use. It can be divided into three steps: 1.2.1 Sampling
1. Write the details on the enclosed label and stick the label on the tube (do not forget the date of 2. Unscrew the cap of the container and take out the slide without touching the agar surface.
3. If a glass beaker is used to obtain the sample, ensure that the sample is taken from a point with good mixing so as to obtain a representative result.
If sampling directly in the circulating system, the slide should be dipped into the liquid to be tested atan accessible point or held carefully in the stream of liquid. If the liquid splashes at high pressure,take care that the agar does not become detached from the slide.
(When testing surfaces and solid media, both sides must be pressed onto the surface. In the case ofplaces that are difficult to access, a sterile swab may also be used, which is then streaked out ontothe agar.) The slide should be in contact with the liquid that is to be tested for approx. 5 – 10 seconds. Both agarsides of the slide must be completely covered.
4. Allow excess liquid to drip off the slide (a small amount of liquid that settles at the bottom of the tube has no effect on the result). Recommendation: tap the lower edge of the slide on clean filter paper.
5. Replace the slide in the tube and seal the tube.
1.2.2 Incubation
The optimum incubation temperature is generally that which corresponds to the practical conditions. Forcoolants a temperature range of 25-30°C has proved optimum. Incubation should if possible be carriedout in an incubator (but is also possible at room temperature).
After 24 – 48 hours the result can be read off from the TTC agar (bacteria).
Room temperature: 2 – 4 days.
For slow-growing organisms a check should be made after a further 48 hours.
Yeasts and moulds grow after incubation for 72 hours.
Room temperature: 4 – 7 days.
After incubation, the density of the colonies on the agar surfaces should be compared with the evaluationchart. The evaluation is carried out with the tubes sealed.
Schülke & Mayr
1.2.3 Interpretation of the results
Normally an evaluation chart is enclosed with the dipslides. It shows different forms of microorganismcolony formation on the dipslides. The nature and number of colonies formed corresponds to the degreeof microbial contamination. The microbial contamination is given in cfu/ml (cfu = colony-forming unit).
1. For evaluation of the result the colony growth of the incubated samples on each side of the agar is compared with the pictures on the evaluation chart.
2. The picture that is most like the colony density on the agar slide is taken as the result.
The majority of bacteria grow to form red colonies. Growth of colourless colonies is also possible.
Note! These colourless colonies must also be taken into account.
Important:
If colourless colonies of bacteria grow completely together, it can look as if the agar slide is free ofmicroorganisms. This then gives the impression of a negative finding. In unclear cases it is best to letlight reflect onto the agar slide and compare its appearance with that of an unused slide.
When examining the incubated agar slides, it is the number of colonies that have grown that isimportant, not the size of the colonies. Large-area colonies can give the wrong impression of heavycontamination (very dense growth).
4. If the microbial growth on the agar slide clearly lies between two comparison values, the values of the pictures between which the number of colonies on the slide falls is taken as the result (e.g. 104 – 105).
On the Rose Bengal agar both pure mould or yeast growth or mixed growth of moulds and yeasts ispossible.
Moulds form woolly colonies made of up of individual threads or thread aggregates.
The evaluation is given as follows: slight contamination (+), moderate contamination (++), heavycontamination (+++).
1.2.4 Disposal of incubated slides
After the dipslides have been used, the possibility that pathogenic organisms have also colonised thenutrient media cannot be ruled out. There is therefore the question of safe disposal. In principle there aretwo possibilities: The safest and cleanest method is autoclaving. For this, the used nutrient media are packed into either aspecial drum or an autoclave bag (cleanest solution) and then heated in the closed autoclave to 121°C for15-20 minutes.
After cooling, the remains can then be disposed of with domestic waste.
However, autoclaving is only suitable for industrial operations that have a large number of samples.
For smaller operations chemical disinfection is recommended. For this, the used dipslides are immersedfor 18 h in a 10 % disinfection solution (Lyso 3025, Buraton 10 F, Parmetol DF 35, etc.), and thendisposed of with the domestic waste.

Source: http://www.gianniberti.it/Editoriali/sm/grotan/use_of_dip-slides.pdf