Examination of bacteria using a microscope is not sufficient to be able to reach a definitive identification – although some types of wine bacteria do have a characteristic appearance. More detail about the specific types of bacteria found in wines can be found in the microbiological hazes and deposits section of the Solutions website. There are also microscope photos of a number of types of bacteria commonly found in wine in the picture gallery section of the site.
There are, however, some tests that can be applied to isolated bacteria, to gain further information about them. The tests most commonly used are gram staining and the catalase test. These tests on bacteria isolated from wineare sufficient to allow the bacteria to be identified as lactic acid bacteria (LAB) or acetic acid bacteria (AAB). Lactic acid bacteria are gram-positive and catalase negative whereas AAB and Bacillus are gram negative and usually catalase positive although some Acetobacter species are catalase negative. The presumptive identity of the LAB can be confirmed by growing them in a liquid medium containing glucose and fructose but no malic acid and testing for the presence of lactic acid.
To identify the LAB isolates to genus level phase contrast microscopy is useful. Oenococcus oeni cells, as isolated from wine, can occur singly, in pairs or in chains of varying length depending on the strain. They have a spherical cell morphology that often tends to lenticular and also occur as diplococci often giving them the appearance of short rods rather than coccoid shaped cells. A mucoid coating on the cells may also obscure the connection between adjoining cells further adding to the confusion. Often it is useful to slowly focus through the cells moving the objective up and down. Then it is often possible to see the constriction that occurs between adjoining Oenococcus cells. This constriction is not observed when examining the rod-shaped Lactobacillus cells. Under phase contrast microscopy Pediococci are spherical in shape and occur in pairs or in a characteristic tetrad formation that is not seen in any other wine isolated bacteria. They never form the chains that are characteristic of Oenococcus. Lactobacilli, as their name suggests, are rod-shaped organisms that can occur singly, in pairs or in chains of variable length depending on growth conditions and strain characteristics.
Gram staining
Gram staining is one of the most widespread methods used in bacterial identification. It separates bacteria into two fundamentally different groups based on cell wall characteristics. Cells are initially stained with crystal violet and fixed with iodine. The reaction is then neutralised, decolourised and then counterstained with safranine. The preparation is then examined directly using an oil immersion 100X objective with bright field illumination. Phase contrast illumination is not recommended when observation of colour is important.
Gram positive bacteria, which have a peptidoglycan rich cell wall, have a high affinity for the violet stain and retain it even after an alcohol rinse. They appear dark purple to brown after the staining procedure. Gram negative cells have a low affinity for the violet stain , which is rinsed out with alcohol. When counterstained with the safranine stain they appear bright pinks to red.
Generally only acetic acid bacteria, lactic acid bacteria and some bacillus species are able to survive and grow in wine. Lactic acid bacteria belonging to the genera Lactobacillus, Pediococcus and Oenococcus and the Bacillus species are all gram positive while the acetic acid bacteria, Acetobacter and Gluconobacter species are gram negative.
Although it is possible to make all the reagents required for gram staining most wine laboratories, even larger ones will probably find it more convenient and economical to purchase prepackaged gram staining kits available from many scientific suppliers.
These come with specific instruction on usage. Many recommend that cultures should be fresh and 24 hours is often recommended as the age of the culture to be used. Wine bacteria are far slower growing than human pathogens and therefore take far longer to grow to a visible colony size. In particular Oenococcus oenos often requires 7-10 days to grow to a colony size of approximately 1 mm. Gram staining should be done as soon as colonies attain a size of about 0.5 -1.0 mm.
When gram staining the instructions provided by the manufacturer should be closely followed. The procedure is quite messy so work over a sink and wear appropriate clothing.Use a slide holder or rack over the sink. This can be a pair of glass rods spanning the sink at a distance apart that allows the slides to rest on both rods. Blue tack the rods to the sink or connect them with short lengths of rubber or silicone tubing. Various propriety racks are also available. Ensure that all reagents required for the procedure are readily at hand before beginning the staining.
Procedure
- Step one requires the preparation of a bacterial smear. Its purpose is to fix the bacteria to the slide and prevent the sample being lost during he subsequent staining procedure. It can be prepared from either a liquid or solid medium Place one to two loopfuls of liquid culture or a small portion of a colony from a solid medium onto a slide. If using a colony from solid medium a droplet of water to the cells if using a liquid culture add none. Use an inoculating loop to spread the cell suspension over the slide until it covers a third to a half of the slide and there is a uniform thin film covering the slide. Gently warm the slide above a bunsen flame with 2-3 quick passes, smear side up until the preparation is dry. DO NOT EXPOSE THE PREPARATION TO DIRECT HEAT FROM THE FLAME. The slide is now ready for staining.
- Place the dried smear preparation on the slide rack and flood the entire slide with crystal violet and let it stand for 60 seconds then gently wash the slide with deionised water from a water bottle for 5 seconds. At this point the specimen should appear blue violet to the naked eye.
- Flood the slide with the iodine solution and allow it to stand for 60 seconds also, then gently rinse with water from a water bottle for 5 seconds then proceed immediately to the next step.
- Wash the slide gently with the decolourising ethanol by dropwise additionuntil no further blue-violet colour is rinsed from the slide. Too little rinsing and a false positive reading may result, too much rinsing and a false negative may result. For this reason it is very important to gram stain known gram positive and gram negative preparations at the same time as the unknown.
- Flood the slide with the safranine counterstain and let it stand for 60 seconds then rinse gently with water for 5 seconds as before. The gram-negative organisms will take up the safranine and appear pink to red whereas the gram-positive organisms will incorporate little or none of the counterstain and remain violet blue or brown colour.
- Allow the slide to air dry then observe under an oil immersion lens (100X) without a coverslip using brightfield illumination.
Catalase test
The catalase test is an important test because it helps to distinguish between different groups of organisms. In winemaking it allows one to distinguish between lactic acid bacteria which are catalase negative and acetic acid bacteria which are catalase positive. These are the two main groups of bacteria found in wine. Occasionally some bacillus species are also found.
Catalase is an enzyme that catalyses the breakdown of hydrogen peroxide to water and oxygen. Hydrogen peroxide is a by-product that develops due to continuous aerobic metabolism. It is harmful to both aerobic and anaerobic organisms and its breakdown is important for a cells survival.
Procedure
Prepare a fresh solution of 3% hydrogen peroxide from a 30% stock solution. To a colony on a plate add a small drop of hydrogen peroxide and observe if any bubbles (oxygen) form around the colony. Alternately place a small drop of hydrogen peroxide on a microscope slide then using an inoculating loop pick up a colony from the plate and emulsify it in the drop of H2O2.
- Positive resultBubbles will appear around the treated colony on the plate or from the emulsified colony
- Negative resultNo bubbles will appear around the treated colony on the plate or from the emulsified colony.
It is very important to ensure that both positive and negative control organisms are used along with the unknown to ensure the method is working as expected.
However, microscopic examination does not usually give sufficient information to identify the microorganisms responsible for an instability.
For example, most yeast found in wine are ovoid to spherical in shape, so examination of them under the microscope does not enable species identification.