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Carola Fortelius ERASMUS intensive course Tikkurila, 1st of June 2010 • Microbes – who are they? • Microbes – how to work with them • Cultivation of microbes • Factors affecting microbial growth • How to control microbial growth in restoration work Intoduction to Microbiology What do you know about nothing ? -------------------------------------------- a lot Intoduction to Microbiology

Where do we meet them in our Intoduction to Microbiology Pictures of different kinds of micro- Bacterial cells (E.coli) and colonies of Salmonella bacteria Paramecium, eng. Different algae types Slipper Animalcule, Intoduction to Microbiology Intoduction to Microbiology Classification of living organisms Micro-organisms • protozoon Common properties of micro- • microbial cells are small, ca 0.01 - 10 μm (human eye can distinguish >100 μm) • undifferentiatied• mostly unicellular (mold and other fungi • usually fast-growing • large surface area in realation to their weight Intoduction to Microbiology Common properties of micro- • elastic, flexible, resilient (=> hard to break) • often very modest with the nutritional • often versatile metabolism • genetical formability • many micro-organisms can be exploited Intoduction to Microbiology Comparative sequence analysis of 16S RNA molecules has
elucidated that life on Earth is of 3 primary lineages,
referred to as domains: (i) Eukarya (ii) Bacteria (iii)Archaea
Intoduction to Microbiology • Procaryotic organisms can be classified according to many different properties: – shape (rod, cocci, tetraed, chain, etc) – composition of cell membrane (Gram + and Gram -) – O metabolism – other metabolic reactions Intoduction to Microbiology – energy source(s) – formation/absence of pigments – chemical composition of the lipids – pathogen or not – resistance to different antibiotics – symbiotic relationships – living environment Intoduction to Microbiology 5007 species of bacteria, or in other words, around 1 % of all until now identified excisting species Examples of bacterial classification Class, genus and species: • Bacilli Bacillus (Bacillus subtilis), Listeria, Streptococcus (Streptococcus aureus), Staphylococcus, Lactobacillus, Lactococcus, etc • Gammaproteobacteria Escherichia (E.coli), Pseudomonas, Salmonella, Yersina, ym. • Actinobacteria ie. Mycobacterium, Streptomyces Intoduction to Microbiology Some common bacterial strains Bacillus subtilis Escherichia coli Note that the magnification is different in different images! Intoduction to Microbiology • Archaea are simple cells that don't have • They are phylogenetically differing from • They are often found at extraordinary and growth places; such as swamps, sediments at the bottom of the ocean, rumen (= 1st stomach of the cow) where the temperature, osmotic pressure, pH, oxygen free or H - atmosphere is extrem • 217 identified species Intoduction to Microbiology Archaea thrive in Microbial sampling in acid mine drainage deep underground in the Richmond Mine, Iron Mountain, Calif. The water is almost as acidic as battery acid, with a pH of about 1. (Paul Wilmes photo) Biogas: Methanogenic archaea are responsible for all biological sources of methane Intoduction to Microbiology • procaryotic and eucaryotic photosyntetic organisms with chlorophyll and other photosyntetic pigments • releasing O2
• Algae utilize photosynthesis (solar energy) to
convert simple inorganic nutrients into more complex organic molecules, or in other words, algae are primary producers of organic matter which animals depend on either directly or indirectly through the food chain Intoduction to Microbiology Algae, cont.
• occur in freshwater, marine water, and damp • lifecycle of algae can be very simple, involving cell division, or complex, involving alternation of generations. • the plant body can be unicellular, colonial, filamentous, siphoneus (?) , or parenchymatous*; but never with roots, stems or leaves • not a natural group because of so many different properties but the name is useful in many contexts * primary tissue of higher plants, composed of thin-walled cells Intoduction to Microbiology Algae, cont.
Respiration is the reverse process of growth in which protoplasm undergoes endogenous decay and/or cell lysis and oxidation. Through respiration and decomposition, organic matter is returned to the simpler inorganic state. During this breakdown oxygen is consumed and carbon dioxide is liberated. Although algae respire oxygen in the presence of sunlight, the amount produced via photosynthesis usually exceeds the amount used during daylight. Intoduction to Microbiology Different kinds of algae (brown, green, red) Ulva fasciata Intoduction to Microbiology Critical nutrients to algal growth usually include phosphorus or nitrogen (though C, Si and light limitations may play a role) cyanobateria produce toxical compounds Cyanobateria ("blue algae", not a true algae) Intoduction to Microbiology Intoduction to Microbiology Biofilms are formed of microbes, growing surface. The film is often very thin an Intoduction to Microbiology Biofilms, cont.
The microbes existing in biofilms are less sensitive to desinfectants and other chemicals than free living cells. They can also stand heat, dryness and radiation better. -> the biofilms are very (very) hard to destry! In nature most microbes exist in biofilms.
Intoduction to Microbiology Biofilms, cont.
Biofilm microbes, mainly bacteria, attach to surfaces and produce free slime or slime capsules around theselves. the slime consists mainy of polysaccharides. The slime protects the cells during unfavourable environmental circumstances and biological and chemical attacks.
Intoduction to Microbiology Biofilms, cont.
The formation of biofilms is a big problem in drainpipes and many industrial processes, for example at the 'wet end' of paper machines and in food industry If toxic compounds are not effective, one solution to the biofilm problem can be the use of other, competing microbes, which do not produce slime. This kind of solution is called competitive Intoduction to Microbiology Molds and mushrooms (Fungi) Molds can be found almost everywhere because they are very modest in their nutrial demands Soil is the most common living environment They are eukaryotic, and can have both sexual and non-sexual reproduction; the non-sexual reproduction via spores Vegetative growth takes part as mycelium Mushrooms produce terrestial fruiting bodies (above Intoduction to Microbiology – Ascomycetes– Basidiomycetes– Phycomycetes– Fungi Imperfecti Fungi with fruiting bodies large enough to be readily visible will usually belong to one of two main groups. The Basidiomycetes
or the Ascomycetes. The main difference between these two
groups is in the way in which they produce their microscopic
spores.
Intoduction to Microbiology Fungi with spores produced inside a sac called an ascus. Each ascus usually contains 8 spores (sometimes four, - depending on the species).
Intoduction to Microbiology Fungi with spores produced externally, on specialised cells called basidia.
Typically, there are 4 spores per basidium, although this varies from 1 to many, depending on the species.
Intoduction to Microbiology Fungi Imperfecti Molds, that have never been found to have a sexual form but whereupon it is found, the Fungi Imperfecti- strains are classified afresh, either as Ascomycetes or Basidiomycetes Intoduction to Microbiology Intoduction to Microbiology Intoduction to Microbiology Intoduction to Microbiology Aseptical working methods • By handling your microbial samples and cultivations in the right way (=aseptically) you yourself will not be infected, ……….neither will your samples be contaminated, but stay clean! Intoduction to Microbiology The aseptic technique is a method of preventing contamination as well as a method of sterilization. It means using common sense when handling your cultures, such as not placing your hand over an open container, not touching a pipet to any surface where it could pick up a contaminant, and keeping all open cultures, media bottles, etc., inside the sterile air containment area. Hospitals use the aseptic technique to prevent infection during surgery. It can also be used in our homes by simply washing our hands before preparing food as well as washing dishes in the hot water of our dishwashers. Intoduction to Microbiology Joseph Lister, who introduced the use of carbolic acid antiseptics in 1865 William Halstead, who advocated the use of surgical gloves in 1898 Intoduction to Microbiology Intoduction to Microbiology Aseptic techniques Aseptic techniques are ways of working that seek to eliminate cross contamination of people and materials. The two main points of aseptic technique is (1) to protect the person doing the work and (2) to protect the work itself, to avoid contaminants. To ensure that this happens, you should begin and end the day with a clean work area and clean hands, and also clean during the day whenever you enter or leave the laboratory. Intoduction to Microbiology Bunsen flame to heat up the vessels, tubs, inoculation loops, etc, Chemicals to clean the surfaces (desinfectants), Special rooms for handling the microbes, samtimes even clean rooms Laminar safety cabinet, UV-lamps for illumination during night time, sometimes addition of antibiotics to the growth Intoduction to Microbiology Intoduction to Microbiology Some practical advice when working in the microbiology lab • At the beginning of work, you use cleaning solutions to disinfect work benches and equipment surfaces that might be sources of contamination • Make sure that you thoroughly clean your hands, wear clean personal protective equipment and keep them clean throughout the day.
• Sterilise all media to be used for growing microorganisms in • Make sure that you do not transfer microbes to or from other areas of the workplace, including lunchrooms and toilets. No items from the laboratory should be taken into other areas such as lunchrooms, etc. Intoduction to Microbiology Some practical advice when working in the microbiology lab • Put all wastes and equipment used to make media into their proper containers for sterilisation • Make sure that Log Books, result sheets, manuals, pens and other work items are not placed on preparation benches, where they can be contaminated and become a source of infection to you and others • Attend to all spills, even small ones, straight away. It is not the size of the spill or the amount of mess that it makes that is the important thing with microorganisms. All spills in a microbiological laboratory are potentially hazardous sources of infection Intoduction to Microbiology When you finish your work: • disinfect all work surfaces • discard protective clothing • wash and/or disinfect your hands Intoduction to Microbiology Autoclave for sterilization of microbial devices Intoduction to Microbiology Intoduction to Microbiology • Microbes are tranfered from one cultivation container/dish/etc with a sterile inoculation loop or pipette or directly poured • inoculation work is performed in a clean laminar airflow safety cabinet • the microbe to be cultivated is often in pure form (not many species mixed together) = a 'pure culture' • growth conditions are regulated Intoduction to Microbiology sterile inoculation loops Intoduction to Microbiology Working in a laminar safety cabinet Intoduction to Microbiology Cultivation techniques 1. Cultivation on a solid surface A mixture of suitable nutrients in water + powder of Agar-agar-algae is heated up to make a gelatinous solid medium. This algae is very resistant to biodegradation so the microbes cannot 'eat' it and it stays uneffected in the growth. When growing microbes on agar media one can regulate only the temperature and surrounding gas atmosphere Intoduction to Microbiology Intoduction to Microbiology Intoduction to Microbiology The origin of the microbe you want to cultivate can be, 1. A pure and identified cultivation bought from some commercial collection 2. A single colony from a mixed own sample (then you often have to do several dilutions and surface strikes bofore you ge a pure cultivation) 3. Any kind of a microbial mixture - not very often used as such (you usually try to get them one by one) Intoduction to Microbiology Intoduction to Microbiology 2. Commercial 'Ready to use' agar plates Intoduction to Microbiology Intoduction to Microbiology Things affecting microbial Intoduction to Microbiology Things affecting microbial growth: 4. Osmotic pressure (salt concentration) Intoduction to Microbiology The nutrients for cultivation of different microbes For a solid medium you usually add 1,5% (15 g/l) agar to the nutrient broth Examples of common media for cultivation of bacteria: Plate count (TYG-) agar meat extract 3 g/l yeast extract 3 g/l Nutrient broth + 15 g/l agar Intoduction to Microbiology Nutrients for cultivation Examples of mold media: Potato dexrose agar sacharose 30 g/l dried potato flour 200 g/l KCl 0,5 g/l FeSO 0,01 g/l Intoduction to Microbiology Intoduction to Microbiology Environmental factors affecting microbial growth: The microbes are divided into three main groups according to their optimal growth temperature: - psychrophile -5 - 20 C - mesophile 10 - 45 C - termophile 40 C -> Intoduction to Microbiology Environmental factors affecting microbial growth: The microbes are divided into three main groups according to their optimal growth pH value: - acidophile 0 - 7 -neutralophile 3 - 10 - alkalophile 7 - 14 Intoduction to Microbiology Environmental factors affecting microbial growth: Salt concentration 2. The salt concentration in a hypertonic solution is higher
than in the cell and the cell will
"bleed water" and dry
1. The salt concentration in an isotonic solution is equal 3.The salt concentration in a
to the concentration a the hypotonic solution is lower
than in the cell and the cell will "swell like a balloon" Intoduction to Microbiology Intoduction to Microbiology Control of biological growth as a restoration method Control of biological growth as a method for conservation Before the treatment method is chosen you should ask the following questions: - Is the treatment necessary? - Will the artefact stand the treatment? - What will be the result of the treatment? - How and when will it be done? - How often do you have to repeat the treatment? - Is the treatment safe for the worker, is there any better method? Intoduction to Microbiology Intoduction to Microbiology Mechanical methods • Remove the microbial growth mechanically either by hand or with some tool (ie. with a scalpel, spatel, saw, hair dryer, • The results are usually not stable • Can cause damage to the object (ie. root removal, forceful • To remove lichen from stone either strong soda or 5% ammonia can be used as a pretreatment to make the lichen Intoduction to Microbiology Examples of methods in use: high frequence electrical current low frequence electrical current low pressure (vacuum) light traps (for insects) Intoduction to Microbiology • max effect 230 – 275 nm • different action depending on the growth phase • RH <60% (humidity low), otherwise no effect at all • usually no very effective • can destroy your artefact! Intoduction to Microbiology • ok for insects, molds can stand it • can be used for paper and wood but paper quality can be poorer if you repeat the • 500 Gy is the minimum dose • does not affect lead and tempera paints • no secondary radiation or remains Intoduction to Microbiology High and low frequency currents: • high frequencies kill wood insects (ie. Anobiidae), but metals make the treatment ineffective • high frequency sound drives away birds (2000-3500 V and 0,5 mA); no harm to humans and animals Intoduction to Microbiology • By using hot air or steam • books: 95 C, RH 40%, t= 4 hours • Old textiles should not be treated with dry air! Intoduction to Microbiology Might be possible to use for • some archeological artefacts and • modern textiles, not old ones! Intoduction to Microbiology Biological methods One can use natural phenomenons such as parasitism or antagonism, i.e. viruses or insects that cause some illness, to get rid of the primary problem (plant growth, microbes, birds).
All possible consequences should be taken into account before the treatment is started! Intoduction to Microbiology Remember always to check the properties of the chemical you intend to use, how to protect yourself and other safety regulations, what is the first aid if something happens. This information is found on the safety sheet that you can ask for when you buy the Intoduction to Microbiology Biochemical methods This group consists of treatment with ie. streptomysin ja penicillin have been used to treat stone and wall paintings, different antibiotics ais effective on different other antibiotics are pimafusin, kanamysin, econazole and Intoduction to Microbiology - enzymes can work as biocides (they catalyze the break down of - trypsin is a protease-enzyme that catalyzes the break down of - the enzymes can only function during optimal circumstances (pH, temperature, substrate concentration etc) => the reaction does not always take place, if the circumstances are not the right Intoduction to Microbiology initially a food preservation product no long term effects "attraction substance" attract specific insects into traps, used in museums Intoduction to Microbiology Thank you for your attention! nothing -----------------------? --------------------- a lot
Intoduction to Microbiology

Source: http://users.evtek.fi/~carolaf/Esitelmat/Erasmus%20Microbiology%201.6.2010-1.pdf