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ISSN 2220-4962 (Print) MicrobeMicrobe ISSN 2220-4970 (Online) Volume 1, Number 10 October 2011

The Magazine for the HunterHunter Enthusiast Microscopist Microscopy Magazine http://www.microbehunter.com

How to find Tardigrades

A Beautiful Slide

Aseptic techniques

Some Terminology

Lichen Microscopy

Diatom Cities

Tardigrade isolation Lichen Microscopy

MicrobeHunter Microscopy Magazine - October 2011 - 1 ABOUT

Microbehunter Microscopy Magazine ANNOUNCEMENT The magazine for the enthusiast microscopist MicrobeHunter Magazine is a non-commercial project. Visit the Forum! Volume 1, Number 10, October 2011 ISSN 2220-4962 (Print) It is now possible to discuss the individual articles ISSN 2220-4970 (Online) of the magazine. Every issue has a separate sub- Download: Microbehunter Microscopy Magazine can be down- forum for discussion. loaded at: http://www.microbehunter.com www.microbehunter.com/forum Print version: The printed version can be ordered at: http://microbehunter.magcloud.com Facebook Publisher and editor: Oliver Kim, Ziegeleistr. 10-3, A-4490 St.Florian, Austria Email: [email protected] Do you have any microscopy links to share? Web: http://www.microbehunter.com Do it here on facebook: Tel.: +43 680 2115051 www.facebook.com/microbehunter Text and image contributions by: Leonardo Balbi Rodney Brightwell Brudersohn (cc) Debivort (cc) Charles E. Guevara Mike Guwak CONTRIBUTE! Paul Hebert (cc) Oliver Kim J. Marqua (cc) Mike Shaw Write for Microbehunter! Anthony W. Thomas Please contribute both articles and pictures. Share your expe- Copyright: By submitting articles and pictures, the authors have confirmed that they are the full copyright owners of the ma- riences, problems and microscopic adventures. If you are a terial. Creative commons and public images are indicat- researcher using , tell the readers what your re- ed with a small text next to the image or in the caption. The search is about. Please contribute, even if you consider your- copyright of all other images is with the author of the article. You self inexperienced. If you are a struggling beginner, tell us are not allowed to distribute this magazine by email, file sharing sites, web sites or by any other means. If you want to have a something about the problems that you encountered. If you copy of this magazine, either order one from Magcloud (see link are an active enthusiast microscopist then share your proj- above) or vistit www.microbehunter.com. ects, experiences and observations. Are you a teacher or lec- turer? Share your microscopic experiences from school or Editorial: Article and image submissions are welcome and should be sent to: [email protected]. university. This magazine is made by an enthusiast microsco- For submission guidelines, consult the website at: pist for other enthusiasts. Let‘s work together to make this http://www.microbehunter.com/submission project a successful one. Please send all contributions to: Disclaimer: Articles that are published in Microbehunter Micros- copy Magazine and the blog do not necessarily reflect the posi- [email protected] tion or opinion of the publisher. The publication of these articles does not constitute an endorsement of views they may express. You must own the copyright of the contributions and you re- Advice provided in Microbehunter Microscopy Magazine is pro- tain the copyright of all submitted articles and pictures. While vided as a service and neither the authors nor the publisher can be held liable and responsible for any errors, omissions or inac- we are not able to pay you for your efforts, we will, of course, curacies, or for any consequences (health, hardware, etc.) aris- give you full credit for your contributions. ing from the use of information of this magazine and the blog (or anything else). Conduct all lab work and (microscopy) hardware Guest Bloggers! Yes, guest blogging is also a possibility. modifications at your own risk and always follow the instructions of the manufacturers. Write microscopy-related blog posts, send them to me and I will publish them on the web site. Naturally, I’ll put a link to your blog. Condition: it must be original content and you must be the copyright holder of the text (obviously). When submit- ting articles, please indicate if you want to have them pub- Front Cover: lished on the blog or in the magazine (or both). Large image: Anthony Thomas Left image: Mike Shaw Middle: Mike Guwak Before submitting anything, please read the submissions Right: Leonardo Balbi page on the website: www.microbehunter.com/submissions.

2 - MicrobeHunter Microscopy Magazine - October 2011 CONTENTS

4 How to find Tardigrades Tardigrades, or water , are cute little creatures that can easily be observed with a stereo microscope. Mike Shaw

8 A Beautiful Microscope Slide Victorian microscope slides are not only nice to look at 4 from outside. Some of the specimens that they carry are at least as interesting to observe. Leonardo Balbi 8

11 Introductory Microscopy Projects for Children and Students Are you searching for simple microscopy projects for classrooms or children? Here is a list of ideas. Oliver Kim

12 Safety issues for Microscopists Safety first! Do you know which can be found in your sample? Oliver Kim

15 Some Lichen Terminology 15 An understanding of basic mycological terminology is necessary to fully understand the anatomy of fungi and . Oliver Kim

16 Microscopic observations of Lichens Lichens are fungi that live symbiotically with a photo synthetic partner. Who would have guessed that lichens can can be parasitically infected by other fungi as well? cc-by-sa Brudersohn cc-by-sa Mike Guwak 20 20 Image Gallery

22 Diatom Cities If your freshwater field microscopy agenda “turns sour” - then it’s time to make some lemonade of the micros copy hike!

Charles E. Guevara

) ( feather cover):Chicken (back puzzle the to Answer Gallus domesticus Gallus

MicrobeHunter Microscopy Magazine - October 2011 - 3 HOW TO Water Bears

Tardigrades, or water bears, are cute little creatures that can easily be observed with a stereo microscope.

Mike Shaw

ardigrades (figure 1) are micro- lection that was a hundred years old. scopic creatures that are a maxi- They’ve been frozen and defrosted, put Tmum of one millimeter in size, under , subjected to very high but usually are found to be about half , and zapped with X-Rays. that size. These are little creatures that They come out alive and well. Just add live in moist lichen, , or leaf litter. water. They are harmless, and cute. Yes The easiest place to start is to find cute. They are often referred to as “Wa- some moist lichen, that the yellow fuzzy ter Bears” because they look like little stuff on a tree in your yard. Lichen bears with six puffy legs, and they have grows on the shady side of the tree Figure 1: Tardigrade from West claws that look like those a grizzly (figures 2-4). Orange, NJ would have. In fact, they have two eyes, Scrape some lichen into a paper bag, a nose and a mouth, kind of like a bear. an ordinary lunch bag, or perhaps an So how do you find them, how long do envelope. Don’t use plastic bags, be- Begin by finding a good tree with they live, what climates do they live in? cause the moisture in there will allow soft fluff that grows on the dark side of Those are some often asked questions. mold to grow, and looking at mold is a the tree. Besides trees, you can also They can live a hundred years, with- different science project! find tardigrades in moss. Go for the soft stand extreme heat and cold, and you The best lichen to collect is not that young moss. In figure 5, you can see find them everywhere. They protect hard crusty kind, shown in the bottom moss in all of its stages of growth, and themselves by going into a type of hi- picture. Sometimes the lichen is green- even some lichen growing with it. Tar- bernation called . They roll ish yellow, the softer, the better. See the digrades like to hide in fluff, so that’s up into a dried little ball, and just stay picture in the bottom middle. This li- dormant, with no sign of whatsoev- chen is fairly good. It is greenish yel- er. Scientists have rehydrated them low and soft. Figures 2-4: Yellow lichen on tree, from a piece of moss in a museum col- fluffier lichen on tree, hard crusty li- chen.

4 - MicrobeHunter Microscopy Magazine - October 2011 Figure 5: Moss in all of its stages. Figure 6: Lichen on brick. what to look for. Simply pull up some figure 6. It’s fine. Tardigrades are sure half inch of water, like a little bowl or clumps of moss and put it in a paper bag. to be in this lichen. water for a hamster. The “dish,” as we Again, use paper bags, not plastic Next, you will need a clear plastic will call it, does not have to be round. bags, when collecting moss and lichen, petri dish, or something like it. You Sprinkle some lichen into it, just a because that allows air to dry out what could use a clear plastic container, like tiny pinch. Remember, this is a micro- you have collected. You do not want the lid from a food container, the top of scopic journey, so a pinch is a ton of mold to grow on your moss or lichen, a little plastic box for cosmetics, a piece material under the microscope. Then and that’s what will happen in a plastic of cut of blister packaging that hangs on add some bottled water, again, just bag that is air tight. So always use the rack in the store. about half in inch. Set aside, and let the paper bags, the kind you get to pack Most small products are blister lichen soak over night. Ideally, you lunch sandwiches in. packed (figure 12), that is packaged in a could use plastic petri dishes, as you can You might even find some lichen on hard plastic shell backed by the color cover these to prevent dust and mold a rock or a brick wall, as shown in cardboard product description. Cut out spores from getting into the water. In a piece of plastic that will hold about a the picture here, you can see that I have

Figure 7: Looking for tardigrades through a compound mi- Figure 8: Looking for tardigrades through an inexpensive croscope dissecting microscope

MicrobeHunter Microscopy Magazine - October 2011 - 5 set up two petri dishes (figure 11), one which I purchased second hand for un- Figure 9 (top left): Petri Dish with Fi- with moss and one with lichen. When der $100.00 on E-Bay. This is a very ber Optic Light you add the water and let it sit, it is high quality Bausch & Lomb dissecting called a “suspension.” microscope, however you don’t need Fighre 10 (top right): Dissecting Mi- You will need a microscope. A dis- anything so fancy. croscope with fiber optic illuminator secting microscope (Figure 8) is best, Nowadays, you get fairly good opti- and cable on left. Tardigrade suspen- but there are some very good amateur cal quality for your money spent. You sion on the base. microscopes available at reasonable can get one from a few places on line prices. Sometimes called a binocular such as Figure 11 (bottom): Petri Dish Sus- stereo scope, it will open up a whole http://www.scientificsonline.com/ pensions of Lichen (left) and Moss new world to you. Here is the one I use, similar to the one shown in figure 8. (right)

6 - MicrobeHunter Microscopy Magazine - October 2011 In fact, higher powers make it more give you what’s called a good working difficult to search through the jungle of distance, or separation from the surface lichen you have created. When ready of the water. to examine the specimen under the mi- Once you have focused on the bot- croscope you need to place a piece of tom of the dish, and the light is stream- black paper under your clear dish or ing sideways across brightly container. Place that on the microscope illuminating the lichen, you can me- stage. In figure 9, you see a petri dish thodically examine the specimen. You with light from the side. Somehow, you are hunting for bears. These are slow will have to mount or set up a narrow moving creatures, which is why they beam of light, from a flashlight or even have been named tardi-grades, or slow- a high intensity desk lamp. Shoot the walkers. What does a tardigrade look beam across the bottom of specimen like? dish. If you Google search, you will see In other words, you do not illumi- many examples. Your Google search nate the lichen from underneath, like would probably lead you to my website you would if you were examining a with loads of pictures showing the vari- glass microscope slide. No. You will ous types of tardigrades found in my have to illuminate the lichen suspension New Jersey Tardigrade Survey: Figure 12: Blister pack. from the side. A horizontal beam makes www.tardigrade.us any tardigrades or other creatures now Looking in your petri dish, don’t be appearing in your specimen dish glow discouraged if you don’t find any at The nice thing is that you do not need white, and they will stand out clearly. first. Sometimes, they can take two or high power to see and observe tardi- Now you have to focus the micro- three days to emerge from their hiberna- grades. scope, at lowest power, on the debris tion. Sometimes you have to look in Your lowest magnification will be that rests on the bottom of the dish. If another sample of lichen or in a sample perfect, as only 15 to 30 times magnifi- you try to use a higher power objective of moss or lichen. Eventually, you will cation is all you need to find a tardi- lens, you will wind up with it under find one. grade. Also, check out what I have in water. You do not want that to happen, There is a saying that the first tardi- my Microscope Store. or you will ruin the optics. So, use the grade you ever find is the hardest to find. lowest power objective, and that will Happy Hunting! Mike Shaw. ■

Daphnia or Simocephalus?

would like to thank Josh Grosse for pointing out a pos- Isible misidentification of a water flea in the September 2011 issue. He wrote: “I'm writing in regard to a possible misidentifi- cation in the September 2011 issue of MicrobeHunter. Here you have some excellent photos of a water flea under the label Daphnia. However these usually have a tail spine, except in the first moult, and a lower fore- head. Based on this, I think this is more likely some other rela- tive, probably Simocephalus.”

Suspected Simocephalus: there is no Daphnia pulex: notice the tail spine. tail spine. Image credit: cc-by-sa Paul Hebert

MicrobeHunter Microscopy Magazine - October 2011 - 7 SLIDES Radiolarians

Victorian microscope slides are not only nice to look at from outside. Some of the specimens that they carry are at least as interesting to observe. Here we have a look at some Radiolaria.

Leonardo Balbi

he paths we walk in this life are This slide caught my attention be- very curious. I have always been cause it was mounted with radiolaria, Tfascinated by science and related which may be the most awesome group subjects, with a very special predilec- of organisms to me. At this point, a tion for biological sciences, so that casual reader might wonder: “but, what when I had to choose a university are radiolaria?” course, I chose . Radiolarians are single-celled mi- While going trough the various cro-organisms, exclusively marine, paths of life, I came to not work in the planktonic, solitary or colonial. Belong- area of my training, but moved to the ing to the class Actinopoda, whose technology and informatics. I can't members differ from other by complain. My current work allows me the presence of rigid pseudopods and a to enjoy biology, and my passion, mi- membrane that surrounds the endo- croscopy, as an engaging hobby. plasm (capsular membrane) separating Acquiring equipment and materials it from the ectoplasm. They are known to fuel this passion, I got from one e-bay in the record through his en- dealer one antique slide, mounted for doskeleton, composed originally by hy- epi-illumination view. I do not know drated silica - opal. exactly who made this slide, but it con- Again, thinking of my inquisitive tains the label from W. Watson & Sons, reader, he now asks: “Okay. I know London (figure 1), undated, but proba- what they are. But what is so special bly made at the end of the nineteenth about them?” - Well - I say - they say a century or early twentieth centuries. (If picture is worth a thousand words. On Figure 1: Label from W. Watson & someone with more knowledge about the following pages are some images of Sons this type of material can send me more this slide, and the beauty of radiolarians information, or even indicate some liter- on it. ature, all feedback is welcome.) ■ Figure 2: General view of slide de- scribe in text (Epson CX4500 scanner).

8 - MicrobeHunter Microscopy Magazine - October 2011 Figure 3: Some specimens on slide (Zeiss SM XX).

Figure 4: Another view of slide (Ortholux I - 5x)

Figure 5: Detailed view of a Nasselarian.

MicrobeHunter Microscopy Magazine - October 2011 - 9 Figure 6: Another field of view (Ortholux I - 5x)

Figure 7: Conical forms (Nassellar- ia) and Spherical forms (Spumellar- ia)

Figure 8: All specimens on this slides are fossil forms from Brabados radio- larite (a rock formed mainly by radio- larians)

10 - MicrobeHunter Microscopy Magazine - October 2011 EDUCATION Things to observe with children

Are you searching for simple microscopy projects for classrooms or children?

Oliver Kim

ere is a list of microscopy ideas fully) develop over the course of a few set of permanent slides with some hair that could be conducted with weeks. Do not let the water rot! samples. Hstudents and children: Fungi from cheese (figures 1 and 2): Coins: Coins collect many scratches Observing dust samples: Students Camembert, Brie, Blue Cheese etc. con- (and dirt) over the years. How can the should collect house-dust and bring it to tain edible molds (not hazardous) and scratches be quantified? Is it possible to class to be observed under the stereo or can be used. This is much safer than predict the age of a coin by looking at compound microscope. Careful, some rotting food and observing the molds. the number of scratches? The year is people may be allergic to dust! Alternatively one can also look at non- imprinted in the coin. Observing sand and samples: filamentous fungi, such as yeast. Blue Observing human cheek cells: This Students should collect sand and soil cheese is inoculated with Penicilium. is a classic, really. Using a cotton swab, samples to be observed under the stereo Vegetables and fruits: The teacher some epithelium cells from the inside of microscope. The different color and tex- cuts the tomatoes and mushrooms in the mouth are collected and transferred ture of the sand grains can be observed. various ways, they can be observed un- to a microscopic slide. Observing textile fibers: Observing der the stereo microscope. Do not eat There are also a few things that one various fibers obtained from clothing the food afterward, you never know better does not observe in a classroom (cotton, polyester, nylon etc.). Different what chemicals were left behind on the setting. Safety, after all, should play a colors and textures become visible un- microscope by previous classes….. high priority. This includes spoiled food der the microscope. Hair samples: Each student donates material and any microogranisms ob- Which printer is the best? Students one hair and then they have to match tained from the human body or . bring in print-outs of different pictures them with the hair left behind on the Blood samples or other body fluids may on different types of paper. The printing “crime site”. This is a playful approach be sources for infection. Polluted water resolution can be observed under the into forensics and gives the observation is also problematic for obvious reasons. stereo microscope. some purpose. Maybe a competition Water from polluted rivers, lakes may Observing water life: A large jar is between different groups is also a nice contain toxic substances and harmful filled with pond water and a little soil. idea. The teacher may have to prepare a microorganisms. ■ and other organisms will (hope-

Figures 1 and 2: Blue cheese. Scratch some of the cells off the cheese and make a wet mount.

MicrobeHunter Microscopy Magazine - October 2011 - 11 ADVICE Laboratory Safety

Safety first! Do you know which microorganisms can be found in your sample?

Oliver Kim

uch has already been said and organisms are alive and depending on Aseptic technique written about the precautions the type of organism, they may pose a Mthat one should take when possible health hazard. The term aseptic technique refers to dealing with organic solvents, fixatives, It it not, and can not be the intention a medical or laboratory procedure that and stains, which are needed for prepar- of this article to give a detailed over- is performed under sterile conditions. ing microscopic specimens. Organic view of the aseptic procedures used in a The aseptic technique fulfils several solvents (such as xylene) can be inhaled microbiology laboratory. I am not going objectives. First, the technique should and many volatiles pass easily through to address the growing of bacterial col- protect the sample under investigation the mucous membranes into the blood. onies on agar petri dishes or the the from contamination. This is of particu- Certain fixatives will react with sub- making of a nutrient broth for the en- lar importance when culturing microor- stances in the cells, where they may richment of . I am also not go- ganisms, as fast-growing contaminants denature and cause a wide ing to address the proper use of an may possibly grow faster than the mi- range of other chemical modifications. inoculation loop and a Bunsen burner croorganism that one is interested in. Stains can be a particular problem, es- for sterile colony transfer. These labora- The sample may thus quickly become pecially if these interact with the DNA tory methods are, in my opinion, too overgrown by unwanted microorgan- of the organisms, as used for making specific for the majority of amateur mi- isms. nuclei visible. In this case the stains croscopists and require a properly While still working in a microbiolo- may be cancer-causing. As a matter of equipped lab and appropriate training. gy lab, I was told that a student working fact, some more traditional substances Such issues can also not be covered in towards his diploma thesis accidentally used in microscopy, such as Hoyer's the little space available. The growth of sub-cultured a contaminant for several mounting medium, contain ingredients (unknown) bacteria on agar plates or months. All of the experimental tests that are addictive and are a controlled liquid culture medium also poses a po- were performed on this contaminant substance and are therefore not freely tential health hazard, as the bacterial and at the end of the thesis work the available. densities can be extremely high, and I obtained data of several months was Much less has been written about generally would be cautious when considered worthless. A quick check of the precautions that one should take working with nutrient media. There are the under the micro- when dealing with the organisms them- also legal issues associated with these scope would have quickly revealed the selves. It is now my objective to address methods, as the legislation of some mix-up. For those of you who were some precautionary measures when countries only permit the enrichment wondering: Luckily I was not the unfor- dealing with organisms that are to be and growth of bacteria for certified lab- tunate student. microscoped. oratories. As a matter of fact, the growth Second, the aseptic technique Amateur microscopy certainly can of unknown bacteria isolated form the should protect oneself from infection by not be considered a high-risk hobby, environment even requires the applica- potentially pathogenic microorganisms. especially when one looks at ready- tion of aspetic methods of an elevated The procedure therefore includes mea- made permanent slides. Here the organ- biohazard level. Readers who are inter- sures that prevent the inhalation of mi- isms in question safely killed and em- ested in these methods should consult croorganism containing aerosols as well bedded in mounting medium. The issue introductory microbiology books, as the prevention of skin contact and starts to look a little different when one which cover these aspects in detail. ingestion. engages in collecting, concentrating and Rather, I would like to place a focus Last, the environment and other possibly even growing microorganisms on the methods that are relevant for people should be protected as well. for microscopic observation. Safety is- microscopists. In particular, I would Proper disposal of petri dishes and mi- sues like this are not only relevant to like to address the making of a hay croorganism-containing sample materi- amateur microscopists, but also for infusion, the observation of pond water als is therefore necessary and often also teachers who want to conduct basic mi- as well as the observation of molds and required by law. crobiological and microscopic work in other fungi. a school laboratory. In this case the

12 - MicrobeHunter Microscopy Magazine - October 2011 Risk Assessment anobacteria, for example, are known to film will start to form on the water cause eye irritations or allergies. surface. This film is teeming with bacte- The dangers of contacting an infec- ria. In the presence of , the num- tion depend on several aspects: Hay infusion issues ber of bacteria may decrease over time, Immune status of the person: The and a progression of different organ- weaker the immune system of the per- A hay infusion is a culture medium isms can be observed. son, the higher the chance of contacting which is commonly used to grow pro- One should be aware that unknown an infection. For this reason, only han- tists, such as the well-known Parame- (and therefore potentially pathogenic) dle unknown bacteria if you are healthy cium, for microscopic observation. Hay microorganisms may also start to grow and have no immune system problems. infusions have been popular since the in the hay infusion. The boiling process Infectivity of the organism: Some beginning days of microscopy and are does not necessarily kill all of the mi- pathogens can be infective at a low still a popular way of obtaining proto- croorganisms present on the hay. It is concentration, others require a higher zoa for educational uses in schools and not uncommon to find heat-resistant concentration. Keep the concentration universities. spores of Bacillus and Clostridium on of the microorganisms low. There are two ways in which a hay the hay. After the cooling of the infu- Density of the organism: The high- infusion can be made. A handful of hay sion, these spores may start to germi- er the density, the higher the chance that is boiled with water to extract nutrients, nate giving rise to live, possibly a critical level of the microorganism is which serve as a food source for the pathogenic, bacteria. The fact is, that reached to cause infection. Just as microorganisms. The obtained culture you simply do not know what you are above, keep the density of the microor- medium must then be inoculated with growing and appropriate safety precau- ganisms low and only grow them if it is the microorganisms that one wants to tions should be taken. not possible to observe them from natu- enrich. Pond water containing ciliates, It is not possible to determine the ral samples. for example, can be used. Alternatively pathogenicity of bacteria by microscop- Mode of transmission: Different one can try to enrich the microorgan- ic observation. A range of biochemical pathogens prefer a different method of isms that can be naturally found on the and genetic tests are necessary. The transmission. Certain pathogens, for ex- hay. In this case the hay-water mixture enthusiast microscopist should there- ample, are transmitted over the air, oth- is not boiled, but simply left standing fore treat such a hay infusion with ut- ers over water and still others over food. for 24-48 hours. A thin iridescent bio- most care. Do not ingest the hay Others require insect vectors for trans- mission. Most microorganisms are harmless, Figures 1 and 2: Two kinds of biofilms. In both cases unidentified microorgan- but one never knows what substances isms are present. I would assume that the left biofilm (bottom side of a shower they are producing when grown at a stopper) is potentially more dangerous (possible accumulation of bacteria that higher concentration. Certain Cy- were washed off from the human skin). The water with the green algae (right) was never in contact with humans and is therefore possibly safer, but one never knows.

MicrobeHunter Microscopy Magazine - October 2011 - 13 infusion, avoid skin contact (especially may be close to agricultural areas and diluted when you add it to liquid culture if there are open wounds), do not inhale there is the possibility for manure to run medium, losing its efficiency. the aerosols and prevent spills. General- into the ponds. Avoid aerosolization: Some micro- ly avoid contact of the liquid with mu- organisms spread over air. Avoid spill- cous membranes, including the eye. Molds age of the culture medium and carefully Also make sure that the hay is clean and add the disinfectant to the medium be- has not been in contact with excrements Molds can be easily grown by treat- fore disposal, avoiding splattering of of animals. You may otherwise enrich ing an appropriate substrate (such as the liquid. bacteria from the 's digestive sys- bread) with a soil-water suspension. Keep bacterial counts low: Make tem. If a spillage or skin contact has Fungi will start to grow and release sure that the sample (such as a hay occurred, then use 70% ethanol for dis- spores into the air. These spores may infusion) contains many ciliates that infection (mix 7 parts of alcohol with 3 not only contaminate other types of consume the bacteria. Keep the level of parts of water). A higher concentration food in the household, but may also be nutrients low to avoid too many bacteria of alcohol may actually have a lower responsible for allergic reactions when from forming and ensure that the medi- disinfection efficiency. inhaled. Many types of mold also pro- um has sufficient oxygen supply for the Do not simply flush the hay infusion duce potent toxins, which are capable of ciliates to grow. down the toilet. This may cause aerosol causing severe health problems. Do not use polluted water: Dirty formation. Add chlorine bleach to the Prevent the spreading of spores by and polluted water can contain contains infusion and allow the substance to keeping the container with the mold many bacteria and a lower count of the work for a few hours. Some people may closed and avoid air currents which may more interesting ciliates. If the water be concerned that the bleach will then distribute the spores. sample was isolated from a stream that also find its way into the waste water, If you want to investigate molds for came in contact with household waste which is not very environmentally educational purposes, then I would sug- water, then it may be possible that friendly. I would agree, but have no gest that you try to first use edible pathogenic enterobacteria are present. solution to this issue. Be aware that the molds, as can be found on foods, such Do not decompose food: Some addition of 70% ethanol to the infusion as cheeses. teachers like to decompose food to will dramatically lower the concentra- demonstrate the spoiling process to tion of the alcohol. You can add con- General Advice children. Be aware that Clostridium centrated alcohol to the infusion but this perfringens may be found on spoiled is a cost issue (and the glass jar may not Here is some general advice when han- meat or poultry. This bacterium can be large enough). dling samples that contain microorgan- cause food-borne illnesses. Personally, isms. I would not use microorganisms from Pond water safety Open wounds: Do not handle mi- spoiled food for educational microsco- croorganism-containing media if you py. I would resort to much safer and Even pond water may be the source have open wounds or cuts in your skin. easily available bacteria and fungi. of some unexpected surprises. I recently Intact skin can be considered as a very These can be isolated from fresh cheese, introduced mosquito larvae into my effective physical barrier against infec- or example. household this way. The mosquitoes tion and open wounds can be problem- Do not culture bacteria obtained caused me quite some irritation at night. atic. from humans: In particular, do not Be aware that keeping a jar of standing Disinfection: Disinfect hands and inoculate growth medium with bacteria water may even be illegal in countries surfaces with 70% ethanol. More con- from the skin. You may be growing with Malaria, which can be spread by centrated ethanol may actually work Staphylococcus, otherwise. certain mosquitos. less efficiently in killing microorgan- Keep petri dishes closed: This min- Other issues relate to the water qual- isms. imizes the risk of accidentally touching ity of the pond water, may or may not Disposal: Autoclave the used cul- the agar surface, which may be covered be very high. Decomposing animals ture medium at 120°C for 30 minutes. by bacterial colonies. Generally spe- close to the sampling site can give rise This should also be able to kill spores. king, I do not recommend the growth of to microorganisms that one does not If you do not have an autoclave avail- unknown bacteria in petri dishes by want to have in the household. able, then cover the petri-dishes or cul- people who do not have basic microbio- Ponds which are clean enough for ture medium with chlorine bleach. logical training in aseptic technique. swimming should not be problematic, Allow sufficient time for these sub- The bacterial concentrations are simply there are rare cases, where people did stances to work. When you add bleach, too high to be safe. get infected by certain protozoa, howev- be aware that this is a corrosive sub- What is the take-home message? A er. Water samples from ponds which are stance when concentrated. Eye and skin good portion of common sense and ba- rich in (potentially irritating) Cy- contact must really be avoided. Also be sic hygienics will greatly reduce the anobacteria or eutrophicated should be aware that liquid bleach becomes more possibility of you catching an infection handled with more care. Some ponds and will hopefully keep you healthy. ■

14 - MicrobeHunter Microscopy Magazine - October 2011 BACKGROUND Terminology

An understanding of mycological terminology is necessary to fully understand the anatomy of fungi and lichens. Oliver Kim

ive this one a try: The thallus of Thallus: This is the vegetative (non- a lichen is composed of hyphae, sexual) part of a lichen. It is composed Gwhich may be paraplectenchy- of the hyphae of the lichen. matic. The lichen forms either perithe- Apothecia: These are fruiting bodies Figure 3: The cup-shaped apothecia cia or apothecia, depending on the which are (in contrast to the perithecia) can easily be seen on this lichen. species. These reproductive structures open and cup-shaped (Figures 2, 3). Image credit: cc-by-sa by Brudersohn contain a hymenium carrying the asci Perithecia: This is another type of fruit- and the sterile paraphyses. The epithe- ing body of a . The perithecia are cium of the perithecia or apothecia give interspersed between the paraphyses. closed and possess a small hole through a lichen its characteristic color, unless which the spores are released (Figure 1). Soredia: these are structures of lichens it is hyaline. The asci produce spores, used for asexual reproduction. They are Epithecium: This is the thin (often pig- which may also be hyaline and septate. composed of fungal hyphens which are mented) outermost layer of a fruiting Lichens may also form soredia for re- in close association with their photosyn- body. production. thetic partner (green algae or Cy- Did you have problems understand- Hymenium: This is the tissue layer of a anobacteria). fruiting body which produces the asci ing this text? Here is a list of terms that Paraplectenchymatic: This is an ad- (these produce the spores). may help you to shed light into the jective describing the shape of the cells mystery. These terms may also help you Ascus, asci: These are specialized cells composing the hyphae. Paraplectenchy- understand the article on the following of a fungus. They form the spores (typ- matic hyphae are short celled and the pages. ically 8). direction of the hyphae is not apparent. Lichens: These are fungi that live in a Hyaline: A structure that is hyaline The hyphae therefore have a much more symbiotic relationship with a photosyn- allows light to pass through. It is trans- “cellular” appearance and are not as st- thetic organism (algae or cyanobacte- parent. reched ria). Spores: these are the reproductive cells Septate: The spores of some lichens are Hyphae: These are long, filamentous of a fungus. They are formed inside the not individual, but are joined to each structures found in many fungi. They ascii. The spores germinate to form a other. They are then separated by a thin collectively form the mycelium of the new fungus septum. These spores are referred to as fungus. Paraphyses: These are long sterile cells being septate. ■ Fruiting bodies: These are the organs that are located in the perithecia (the of a fungus which form the spores. In fruiting body). They do not colloquial English, the fruiting bodies form spores. The asci are are known as the “mushroom” of a fun- gus. The fruiting bodies contain the ascii, which form the spores.

Figure 1 (left): Closed perithecia with a small opening at the top to release the spores. Image credit: cc-by-sa by J. Marqua

Figure 2 (right): The cup-shaped apothecia. The sterile hyphae are the paraphyses. The asci form the spores Image credit: cc-by-sa by Debivort

MicrobeHunter Microscopy Magazine - October 2011 - 15 OBSERVATIONS Lichen Systematics

Lichens are fungi that live symbiotically with a photosynthetic partner. Who would have guessed that lichens can can be parasitically infected by other fungi as well? Mike Guwak

n this photo essay, I present the stages are pushed downwards and are C: Calcium hypochlorite, P: para-Phe- lichen Lecanora polytropa and its almost not visible. nylendiamin reaction. parasitic partner Cercidospora epi- I Epithecium: The epithecium has a polytropa. Let’s start out with a short brown pigmentation, which can be dis- Are you interested in more pictures of characterization of an uninfected solved in KOH. lichens? Check our Web site at: Lecanora polytropa lichen. Spores: The spores are hyaline, there www.flechtenmikroskopie.de Lecanora polytropa are 8 spores per ascus and they are single celled. The spores measure 9-14 (Hoffm.) Rabenh. This site is by Mike Guwak and Dr. x 5-6,5 µm (Figure 5). Ralf Wagner. Here you can find a vari- Thallus: Grey-white, without soredia. Chemical composition: Thallus K-, C-, ety of different lichens that we collect- Apothecium: Ocher colored and 1-1,5 KC+ slightly yellow, P-. The letters are ed over time. We keep on adding more mm in size. The juvenile stage is char- abbreviations for different chemical re- lichens. acterized by a clearly brighter thallus actions performed on the lichen. A mi- border. The borders of more mature nus sign denotes a negative reaction Figure 1 (bottom): Overview of an un- without color change. K: KOH reaction, infected Lecanora polytropa.

16 - MicrobeHunter Microscopy Magazine - October 2011 Figures 2 and 3: The brown pigments of the epithecium (left) dissolve in the presence of KOH (right). This is a relevant criteri- on for identifying the lichen.

Figure 4 (left): Cross section through the apothecium. Figure 5 (center): An ascus. The arrows point to the apex of the ascus. Figure 6 (right): The spores measure about 10µm across and are transparent.

MicrobeHunter Microscopy Magazine - October 2011 - 17 Cercidospora epipolytropa paraplectenchymatic. Pigment becom- (Mudd.) Arnold ing more intensely blue-green with ad- dition of KOH. Cercidospora epipolytropa is a fungus which is able to infect the lichen Lecan- Spores: 13-23 x 4-8 µm, 1- septated, 8 ora polytropa. The infection can be spores per ascus, boat-shaped. clearly seen in the form of blue-green pigmented spots on the host. The fol- Comments: C. epipolytropa is best lowing description refers to the parasitic identified by ist spore size and its oc- fungus, and not to the host lichen. curence on the host Lecanora polytropa agg. ■ Perithecia: 0,08-0,13 mm wide, em- bedded in the hymenium of the host Figure 1 (top): The lichen L. polytropa Lecanora polytropa infected by C. epipolytropa (the blue- green spots). Wall of perithecia: The lower half contains interwoven, more or less par- Figure 2 (center): The parahyses. allel almost hyaline hyphae, which can These are sterile cells in the fruiting only be vaguely differentiated from the body. host tissue. Figure 3 (bottom): Two spores are Pigmentation: The wall in the upper associated with each other and sepa- area towards the mouth is blue-green to rated by a single septum (“1-sep- blackish pigmented, and becoming tate”).

18 - MicrobeHunter Microscopy Magazine - October 2011 Figures 4-6 (top left, top right and left): The three images show cross sections through C. epipolytropa. In the absence of pigmentation towards the base of the fungus, it is nearly impossible to distinguish the fungal tissue from the one of the host lichen. The pigmentation increases towards the opening at the top.

Figure 7 (bottom left): a single ascus containing 8 spores.

Figure 8 (bottom right): The images shows paraphyses, which are long sterile cells in the spore-forming layer of the fungus.

MicrobeHunter Microscopy Magazine - October 2011 - 19 GALLERY Send images to: [email protected]

Cross section through the root of a Lupinus . Oliver Kim

Wood slice section. This old wood was dug up out of our pond and was aprox. 10-12 feet below ground level. It apears to have charcoal traces in it which are visible lower right. The microscope used was a Bausch and Lomb Dynazoom at 120x. Rodney Brightwell

20 - MicrobeHunter Microscopy Magazine - October 2011 Oxalic Acid under polarized light. Olympus BH2 10x S Plan objective + 1.25x intermediate tube + 2.5x relay tube; Nikon D90, flash Anthony Thomas

MicrobeHunter Microscopy Magazine - October 2011 - 21 OBSERVATIONS Of Diatoms and algae

Figure 1: The flooded microscopy field station. It is too deep to enter the stream here!

If your freshwater field microscopy agenda “turns sour” - then it’s time to make some lemonade of the microscopy hike! Charles E. Guevara

he radical stream flooding rapid- I could not even find my clever cov- plots of variables but also a 'pearl' to ly increased in depth, and also its er-slip collection gadgets, or my strong pursue on my flooded-out microscopy Ttorrential strength. This, natural- magnets in a glass vial. I could only find hike. One page 374 it read: "Another ly, always occurred when I had the free the gauges, which I spiked interesting feature is the frequent asso- time to hike to my field microscopy into the bacterial bloom locations in the ciation of Gomphonema with the basal station (read the article in the August stream, towards the bottom of the shore. cells of Oedogonium, but so far there is 2011 issue about the field station). The Ahh, but a 1964 text in my cluttered no evidence to suggest whether this is a torrent prevented me from reaching my study read: "The Algae". This text tan- casual relationship or not". Now here field station platform, and I could not talized me with a wonderful section was field microscopy hike objective for get to my field location with the purple (Chapter 14, “Freshwater Ecology”, my puppy and me (after I hiked back sulfur bacterial blooms. section “Epiphytes”, pp. 371-374). This home to return my nifty field micro- chapter contained graphs, charts and scope kit). I mustered up my stream

Figure 2: The intact field-microscopy sta- tion (Read the Au- gust 2011 issue) for an explanation.

22 - MicrobeHunter Microscopy Magazine - October 2011 Figure 3 and 4 (top, middle): A stream rock at my work bench, full of epiphytes.

Figure 5 (bottom): The wet-mount preparation. Macroscopically you can see where the epiphytes clus- ter! Yes… yes!!

hike equipment and my non-micro- scope-assistant-puppy, and the usual plastic pail with a specimen jar. I suppose, on an allegorical level, I searched for 'Diatom Cities', but the field microscopy imbued by Dr. Chap- man’s published thoughts was signifi- cant, and motivated me to revisit a seasonal epiphytic assemblage of stream organisms. Please do not enter a raging stream, but rather do enjoy with me this encoun- ter of algae in harsh currents - algae attached to a rock about 7 cm beneath the churning stream surface.

Text sources:

"The Algae", V.J.Chapman, Macmillan & Co LTD/ St Martin's Press, 1964 a wonderful text! Narratives as if you are attending Dr.Chapmans lectures!

"How to Know the Fresh-Water Algae", G.W.Prescott,WM.C.Brown Co.,1954 So useful over the years, thank you, Dr.Prescott!

"Diatoms of North America", William C. Vinyard, Mad River Press, 1979 is brilliant. For example, it states that Diatomella is made of the words Diato- the genus name Cocconeis originates ma and the Latin dimuitive “ella”. The This was very helpful on an area I from Greek, with “cocco” being a berry “di” in Diatoma comes from the Greek know little about, yet constantly en- and “neis” beig feeble. Gomphonema word for “through”, while “tom” means counter. The section on pages 77-79 comes from the Greek “gompho” to cut. So “diatoms”, are Greek rooted titled "Derivations of Generic Names" (wedge-shaped) and “nema” (a thread). as: “cut through”?!! ■

MicrobeHunter Microscopy Magazine - October 2011 - 23 Figures 7, 8 (top): Diatom cities!

Figure 9 (far left): There are different dominant species in different areas of the algal thallus substrate. This be- comes evident in low-power observa- tions.

Figure 10 (left): Gomphonema spe- cies in a bloom of epiphyte assem- blages.

24 - MicrobeHunter Microscopy Magazine - October 2011 Figures 11 and 12 (left) a Diatom City with a desmid (Cosmarian).

Figure 12 (bottom left, right): Cocco- neis species area of epiphyte domi- nance on this algae substrate

Figure 13 (bottom right): Just Like this Chironomid midge larvae, I have traversed a few Diatom Cities. I am now with many questions, I deeply enjoyed this field microscopy hike. Thank you all, whom also enjoyed this field microscopy hike!

MicrobeHunter Microscopy Magazine - October 2011 - 25 What’s this? Answer on page 3.

26 - MicrobeHunter Microscopy Magazine - October 2011