lipscomb, thomas - tem'14 - report

Thomas Lipscomb

Transmission Electron Microscopy and Cell Ultrastructure (BIO4650)

Fall 2014

TEM Micrographs of Homarus americanus

Lobster Digestive Gland and Aerococcusviridans-Infected Gill

1

Introduction

This series of transmission electron microscope micrographs images two organs in Homarus americanus lobster digestive gland and Aerococcus viridans-infected gill Aerococcusviridans-infected gill is being investigated because the lab is trying to understand the pathology of Aerococcus viridans especially how they survive uptake into the lobster by vacuoles and then how the filaments that Aerococcus viridans releases might be the cause of the vacuoles pathologically growing too large Non-infected lobster digestive gland is being investigated by me because it is the control group Aerococcus viridans-infected lobster digestive gland is an experimental group and others are comparing it to the non-infected lobster digestive gland healthy tissue

I hope to see the gill tissue (and even the blood spaces feeding it) containing pathologically large vacuoles containing Aerococcus viridans I hope to see the cuticle of the gill granulocytes mitochondria smooth and rough endoplasmic reticulum and nuclei I hope tosee the lobster digestive gland containing microvilli fixed phagocytes more granulocytes more endoplasmic reticulum more nuclei and more mitochondria

One point of comparison between the tissues of the gill and LDG is that gill is an Aerococcus viridans-infected tissue and the LDG is not The blood spaces in the gill and the blood spaces in the LDG can be compared The gill filaments are similar in structure to the LDGrsquos microvilli but the gill filaments are much larger The cell components of the cells in the gill and the cells in the LDG can be compared endoplasmic reticulum mitochondria nucleus and vesicles I did not find golgi anywhere even though it should be present (Factor 2014)

The lobster digestive gland is located in the midgut (Factor 1995) It is primarily responsible for the synthesis and secretion of digestive enzymes the final digestion of food and absorption of nutrients (Factor 1995) It is also involved in excretion lipid and carbohydrate metabolism storage of lipids and storage of inorganic reserves (Factor 1995) The lobster digestive gland is a pair of large complex glands each with its own duct lying primarily in the cephalothorax (Factor 1995) Each gland is divided into three lobes (Factor 1995) Each lobe has a series of blindly ending digestive tubules that connect to the duct (Factor 1995) The tubules are approximately 100 microm in diameter (Factor 1995) Circular and longitudinal contractile bundles of myofilaments lie between the basement membrane and the tubules (Factor1995) Microvilli increase the surface area of the LDG tubules (Factor 2014) The tubules are interspersed with hepatic arterioles that are covered in fixed phagocytes (Factor 1995) The arterioles supply the tubules with blood (Factor 1995) The fixed phagocytes contain electron-dense granules and are ridged to increase surface area (Factor 1995) The fixed phagocytes are part of the lobsterrsquos immune system by filtering foreign material from the bloodstream and beinga barrier between any germs that might enter from the ingested food in the LDG into the bloodstream (Factor 1995) The fixed phagocytes engulf germs like Aerococcus viridans but Aerococcus viridans somehow survives in the phagocytic vacuoles and over time the phagocytic vacuoles become pathologically large perhaps because of the filaments that Aerococcus viridans secretes (Factor 2014)

The lobster gill contains two blood vessels the branchial stem afferent vessel that carries blood from the gill to the heart and the branchial stem efferent vessel that carries blood away from the heart to the gill (Factor 2014) Those two blood vessels are next to each other and

2

separated by a septum and covered by a circular vessel (Factor 2014) The circular vessel is covered by gill filaments that are long tubes hanging from the circular vessel (Factor 2014) Blood circulates through the gill filaments (Factor 2014)

Our transmission electron microscope uses an electron beam generated from an electron cloud around a heated tungsten filament focused through several apertures and electron lenses to image a thin section (Factor 2014) The section is stained with electron-dense materials so when the electron beam passes through the section the stained parts absorb more electrons than the non-stained parts which creates enough contrast to show detail in the image (Factor 2014)

Materials and Methods

Lobster Digestive Gland (LDG) (No author Appendix 3)

On the first day the samples went through primary fixation gt6 hours-overnight with 3 glutaraldehyde in 02 M NaCac buffer at 4ordmC On the second day the sample was rinsed with buffer four times each for gt15 minutes Then six hours of secondary fixation of the samples with 1 OsO4 in buffer occurred Then gt6 hours-overnight secondary fixation of the samples with another 1 OsO4 in buffer occurred On the third day the sample was rinsed with buffer for gt15 minutes twice and rinsed with distilled water for gt15 minutes four times Then the sample was en bloc stained gt6 hours-overnight with 1 aq uranyl acetate at room temperature On the fourth day the sample was dehydrated for gt15 minutes (each) with 30 ethanol then 50 ethanol then 70 ethanol then 90 ethanol then gt1 hour with 100 ethanol over CuSO4 then gt2-3 hours with 100 ethanol over CuSO4 Then the sample was dehydrated for gt15 minutes (each) with 5050 ethanol and propylene oxide 100 propylene oxide and again with 100 propylene oxide Then the sample was infiltrated for gt2 hours with 5050 propylene oxideand embedding medium and infiltrated again for gt2-3 hours with 100 embedding medium Then the sample was infiltrated for gt6 hours-overnight with 100 embedding medium On the fifth day the sample was embedded for the last time (in a plastic block) cured at room temperature for gt1 hour and polymerized in a 60ordmC oven for 12-24 hours

The block was trimmed down with a razor blade until tissue was reached Then thick sections were made with the LKBCambridge Huxley Microtome to smooth out the block face Then thin sections (100 nm) were made with the LKBCambridge Huxley Microtome and put on copper grids for viewing in the TEM

The TEM was stigmated with reference to a holey grid (Factor 2012) A background image with nothing in the TEMrsquos stage was taken to calibrate the AMT Digital Camera (Factor 2012) The copper grid was inserted into the specimen rod sent through the airlock and placed on the TEMrsquos stage (Factor 2012) When the copper grid with the sample was viewed in the TEM the first condenser lens aperture (300 microm) and second objective aperture (50 microm) were used with the filament at a tension of 80 kV (Factor 2012) But the LDG staining was very faintso micrographs 123-126 used the third objective aperture (30 microm) to improve contrast The micrographs of the samples were recorded using the AMT Digital Camera in quality image modewith autogain on (Factor 2012) Micrographs 1-73 were not well focused Starting with image 74 before each image was taken the AMT Digital Camera was removed and the image was focused with reference to the focusing screen then the beam was spread until it was dim enough for the camera and the camera was reinserted (Factor 2012)

3

Aerococcus viridans-Infected Gill (Surace 2012)

Six live Homarus americanus lobsters were purchased from a local commercial vendor Five lobsters were kept in a 30 gallon tank containing Instant Ocean artificial sea water at 15ordmC and 28 ppt salinity A 50 gallon capacity filter with a foam and charcoal insert filtered the sea water to keep organic debris and chlorine levels to a minimum The tank was in a temperature-controlled room and the lobsters were monitored closely The lobsters were fed a diet of catfish fillets

Dr James Daly of Purchase College provided a suspension of culture grown Aerococcus viridans bacterium diluted to 5106 bacteria per 10 M of inoculant This culture originated fromDr Richard Nilford CT Laboratory of the National marine Fisheries Service who isolated the bacterium from an infected lobster Dr Dalyrsquos culture was injected into the five (experimental) lobsters but not injected into the sixth (control) lobster The times of allowed uptake for the five lobsters were 90 minutes 24 hours three days one week and finally a natural demise after eightdays The control and 90 minute uptake specimens were kept cool and moist in the laboratory before being sacrificed All specimens were anesthetized in a freezer humanely euthanized and dissected to remove tissue samples

Digestive gland tissue gills and blood samples were obtained from all six lobsters and fixed with a Lysine Acetate Ruthenium Red Osmium Tetroxide fixation method following the procedure from the Hammerschmidt et al (2005) experiment Specimens were initially fixed with 20 formaldehyde and 25 glutaraldehyde in 01 M sodium cacodylate buffer The buffer contained 0075 ruthenium red and 0075 M lysine acetate The initial fixation was performed in chilled solutions with a pH of 69 Starting April 18th 2012 samples from the lobsters were placed in this fixative which was then stored in the refrigerator until all lobsters were sacrificed (April 26th 2012) and the remaining steps of the procedure could be carried out

Then a buffer rinse was performed on the specimens in the 01 M sodium cacodylate buffer containing 0075 ruthenium red the specimens were rinsed for a total of three hours The second fixation was then performed with the same fixative used in the initial fixation exceptwithout lysine acetate Samples were then rinsed in the 01 M sodium cacodylate buffer containing 0075 ruthenium red for one hour at room temperature for a total of three rinses The third fixation of the samples was carried out with 10 osmium tetroxide solution in 01 M sodium cacodylate buffer containing 0075 ruthenium red for one hour at ambient room temperature followed by three rinses in 01 M sodium cacodylate buffer containing 0075 ruthenium red at room temperature for one hour A second set of specimen samples were fixed differently for Frank Surace studying the fixed phagocytes but were not used in my project

After fixation and rinses the samples prepared in lysine acetate ruthenium red were dehydrated and embedded The dehydration consisted of ethanol concentrations of 10 30 50 70 90 and two 100 dehydrations over CuSO4 Each step in the series was carried outfor a total of 30 minutes each with exceptions to the 100 dehydrations The first 100 dehydration was carried out for one hour on ice while the second was performed for 24 hours on ice

Specimens were then infiltrated with a solution consisting of a 5050 mixture of 100 ethanol and acetonitrile Two 100 acetonitrile infiltrations each 15 minutes followed

4

Specimen embedding was then performed on tissue with a 5050 mixture of acetonitrile and Araldite 502 ndash Embed 812 plastic resin embedding medium for 15 minutes Tissues were then embedded in 100 plastic resin medium each overnight Specimens were allowed to cure for one hour at room temperature and then taken and put into molds with fresh Araldite 502 ndash Embed 812 plastic resin medium (from Electron Microscopy Sciences Inc) and polymerized in an oven at 60-70ordmC to form blocks for sectioning The resulting blocks were coded with the corresponding vial from which they were taken


The TEM was stigmated with reference to a holey grid (Factor 2012) A background image with nothing in the TEMrsquos stage was taken to calibrate the AMT Digital Camera (Factor 2012) The copper grid was inserted into the specimen rod sent through the airlock and placed on the TEMrsquos stage (Factor 2012) When the copper grid with the sample was viewed in the TEM the first condenser lens aperture (300 microm) and second objective aperture (50 microm) were used with the filament at a tension of 80 kV (Factor 2012) The micrographs of the samples were recorded using the AMT Digital Camera in quality image mode with autogain on (Factor 2012) Micrographs 1-73 were not well focused Starting with image 74 before each image wastaken the AMT Digital Camera was removed and the image was focused with reference to the focusing screen then the beam was spread until it was dim enough for the camera and the camera was reinserted (Factor 2012)

5

Grid Tracking Form

Summary of Micrographs

6

Fig 1 Thomas Lipscomb 068 71000x lobster gill multilamellar vesicle

Fig 2 Thomas Lipscomb 069 22000x lobster gill blood space with A viridans

Fig 3 Thomas Lipscomb 075 11000x lobster gill granulocyte in hemolymph

Fig 4 Thomas Lipscomb 081 71000x lobster gill A viridans with filaments in vacuole

Fig 5 Thomas Lipscomb 083 8900x lobster gill cuticle

Fig 6 Thomas Lipscomb 084 14000x lobster gill nucleus and mitochondria (several cells)

Fig 7 Thomas Lipscomb 090 5600x lobster gill cuticle on left epithelium on right nucleus at bottom left rough ER and mitochondria in middle

Fig 8 Thomas Lipscomb 091 14000x lobster gill mitochondria and rough ER

Fig 9 Thomas Lipscomb 092 56000x lobster gill mitochondria

Fig 10 Thomas Lipscomb 094 110000x lobster gill rough endoplasmic reticulum

Fig 11 Thomas Lipscomb 100 14000x LDG granulocyte

Fig 12 Thomas Lipscomb 105 11000x LDG nucleus between lipid storage vacuoles

Fig 13 Thomas Lipscomb 106 3500x LDG survey view of the LDG epithelium at the top and rest of LDG tissue below

Fig 14 Thomas Lipscomb 110 11000x LDG endoplasmic reticulum

Fig 15 Thomas Lipscomb 111 36000x LDG rough endoplasmic reticulum zoomed 110

Fig 16 Thomas Lipscomb 116 4400x LDG lipid storage vacuoles and microvilli on lower left

Fig 17 Thomas Lipscomb 118 8900x LDG nucleus and granules

Fig 18 Thomas Lipscomb 121 8900x LDG fixed phagocyte

Fig 19 Thomas Lipscomb 124 14000x LDG microvilli

Fig 20 Thomas Lipscomb 126 140000x LDG nucleus upper right nuclear membrane

7

Micrographs with Captions and Observations

8

Fig 1 Thomas Lipscomb 068 Grid D4 lobster gill multilamellar vesicle

I was almost going to not use this image because nobody knew what it was but then I remembered multilamellar vesicles which are vesicles with more than one membrane The cytoplasmic bridge at the bottom of the image suggests that the multilamellar vesicle is undergoing phagocytosis or exocytosis (without seeing which way it was moving I cannot know which way it was going) I cannot distinguish the contents of the multilamellar vesicle There is moderate focus texture I wish that I remembered if this was part of a fixed phagocyte but there was no survey image to remind me what this was a part of I also wish that it had been undergoing phagocytosis of A viridans because Dr Factor wants to find one of those No knife marks and there is good stigmation

httpwwwmikeblaberorgoldwineBCH4053Lecture14Lecture14htm

9

10

Fig 2 Thomas Lipscomb 069 Grid D4 lobster gill blood space with A viridans

These are about six Aerococcus viridans inside of a blood space in the gill The A viridans not only multiply in phagocytic vacuoles but also within the blood of the lobster (Factor 2014) Thefilaments extending from the capsule of the A viridans can be seen I am not sure if the hemolymph is the powdery stuff on the left and right of the image or the smooth part in the middle that surrounds the A viridans If the hemolymph is the smooth space then the blood space ends when the powdery section is reached If the hemolymph is the powdery stuff then thefilaments somehow repel the hemolymph and the edge of the blood space is not visible There is almost no focus texture There is good stigmation

11

12

Fig 3 Thomas Lipscomb 075 Grid D5 lobster gill granulocyte in hemolymph

This is a granulocyte (an immune system cell) in hemolymph The hemolymph is visible in the lower right corner and the edge of the blood space is in the upper left corner The granulocyte contains granules that are the dark spheroids in this image Those granules are secretory vesiclesthat aid the immune system (Wikipedia 2014) The granulocytersquos nucleus and associated endoplasmic reticulum and golgi was not in this plane of section The change in the darkness of the granulocytes seems to indicate that this section is not even in thickness There is hardly any focus texture There is good stigmation There is a tear in the section in the upper right corner ofthe image

13

14

Fig 4 Thomas Lipscomb 081 Grid D5 lobster gill A viridans with filaments in vacuole

This is the highest-mag micrograph of A viridans in the report It was created to best show the filaments that extend from the A viridans capsule This image was of A viridans in a vacuole not another area such as a blood vessel The working hypothesis is that the filaments cause the vacuole to pathologically enlarge but that has not yet been proven (Factor 2014) There is almost no focus texture and that is good for a high mag micrograph The two A viridans are probably together because they can remain a dyad after binary fission (Factor 2014) They are called filaments not extracellular matrix as the caption guesses There are no knife marks There is good stigmation

15

16

Fig 5 Thomas Lipscomb 083 Grid D4 lobster gill cuticle

This is the cuticle of the gill Cuticle is very tough which is why the knife cut it in wavy patterns instead of more smoothly (Factor 2014) There was no time to get another section of gill to try to get a micrograph of cuticle that was less wavy The cuticle is the epithelium that is the barrier between the carapace gill foregut and hindgut of the lobster and the seawater aroundit (Factor 2014) The cuticle is tough because it has a lot of connective fibers and is calcified (Factor 2014) The carapace is the most calcified of the cuticles which makes it the toughest (Factor 2014) There are no knife marks going from bottom left to upper right but there is waviness from the upper left to the lower right There is good stigmation

17

18

Fig 6 Thomas Lipscomb 084 Grid D4 lobster gill nucleus and mitochondria (several cells)

Thin sections are not always in convenient planes For example if the thin section is not perpendicular enough to the membrane then the bilayer looks like a monolayer The problem here appears to be that the section passed through several cells at an awkward angle so only the nucleus (found by the darkness of its heterochromatin) at the top makes sense The rest of the image is a jumble of mitochondria and what may or may not be endoplasmic reticulum or golgi and may or may not be cell membranes There is almost no focus texture but this is a low mag image so that is less apparent There is good stigmation

19

20

Fig 7 Thomas Lipscomb 090 Grid D4 lobster gill cuticle on left epithelium on right nucleus atbottom left rough ER and mitochondria in middle

There is cuticle on the left epithelium on the right a nucleus at the bottom left and rough ER and mitochondria in the middle This is the micrograph that has the biggest collection of organelles in one micrograph But the clarity of the image is not as good as Figure 14 which also has a huge field of organelles but is mostly rough ER There are knife marks going to the right and slightly down There is moderate focus texture There is good stigmation

21

22

Fig 8 Thomas Lipscomb 091 Grid D4 lobster gill mitochondria and rough ER

This is the best survey image of mitochondria in the report It shows the location of the mitochondria in relation to the rough endoplasmic reticulum The mitochondria appear to be evenly distributed around the cell The nucleus may be at the bottom right if those dark areas areheterochromatin There is almost no focus texture but this is a low mag image so that is less apparent The illumination may be off-center The rough ER here is slightly curved but not curved enough to be golgi as the caption guesses it is There is good stigmation

23

24

Fig 9 Thomas Lipscomb 092 Grid D4 lobster gill mitochondria

This is the only high-mag image of mitochondria in the report The inner membrane cristae (inner membrane folds) and outer membrane are clearly visible When taking this image I did not think that I could see anything more if I went to a higher magnification but now I think that if I had I might have seen mitochondrial ribosomes as I had seen ribosomes in the ER I also had thought that the mitochondrial DNA is not densely packed enough to be visible at any magnification but in the lecture page 12 there is a TEM pictures of DNA or RNA that burst froma bacteriophage (Factor 2014) so maybe even unpacked DNA can be seen sometimes There is some focus texture but at this high mag that is pretty much unavoidable The right of the image is slightly brighter than the left either because of a misaligned electron beam or different section thickness There are no knife marks There is good stigmation

25

26

Fig 10 Thomas Lipscomb 094 Grid D4 lobster gill rough endoplasmic reticulum

The gill micrographs need one image that is at least 140000x but all of the 140000x pictures of gill were blurry so this 110000x image is in its place This is the only high-mag image of roughendoplasmic reticulum in the report however Figure 15 somehow shows the ribosomes in the rough endoplasmic reticulum better even though it is only mag 36000x There are periodic blurry dots on these membranes which I assume are ribosomes attached to the membranes The membranes here are very clear but strangely the ribosomes are not they are much more blurry than I expected especially compared to Figure 15 For some reason the right of this image is lighter than the left either because of uneven section thickness or a misaligned electron beam that is not pointing towards the center but towards the right There is some focus texture but at this high mag that is pretty much unavoidable This is rough ER not golgi as the caption guesses There are no knife marks There is good stigmation

27

28

Fig 11 Thomas Lipscomb 100 Grid E3 LDG granulocyte

This is a granulocyte somewhere in the LDG This section appears to have uneven thickness with a light and thin part at the top and a dark and thick part at the bottom The nucleus of the granulocyte is to the right The granulocyte contains granules that are the dark spheroids in this image Those granules are secretory vesicles that aid the immune system (Wikipedia 2014) This is the worst micrograph of the twenty because of the serious thickness unevenness But it isthe only image of a granulocyte in LDG Bad knife marks going up and to the right are visible and unfortunately only one grid (Grid E3) gave me good micrographs of the LDG despite me trying to get better sections after I took this image So bad knife marks will plague Figures 11-20 This image is too low mag for focus texture to appear There is good stigmation

29

30

Fig 12 Thomas Lipscomb 105 Grid E3 LDG nucleus between lipid storage vacuoles

This was an island of darkness in a giant field of light-colored lipid storage vacuoles It could bea blood space with what looks like hemolymph feeding the lipid-storage cells around it but it isstrange that there are no fixed phagocytes visible around the blood space so it is probably not a blood space The dark portion in the center also looks similar to heterochromatin and euchromatin so the dark portion is probably a nucleus The knife marks here are a lot less pronounced than a lot of the other micrographs of Grid E3 but you can still see them they are almost vertical The focus texture is pretty mild and one of the best of all the micrographs There is good stigmation

31

32

Fig 13 Thomas Lipscomb 106 Grid E3 LDG survey view of the LDG epithelium at the top and rest of LDG tissue below

This is a survey image of LGD that tries to capture the morphology of the entire tissue in one image with the epithelium and the basal lamina below it being the horizontal line at the top and the rest of the gill below it Page 411 of the book Biology of the Lobster Homarys americanus was used to label the parts of the tissue in this micrograph (Factor 1995) Just beneath the epithelium should be a band of dark-colored circular muscle The large light-colored parts belowthat are probably blood sinuses The dark colored portions below the circular muscle could be basophilic granulocytes fibroblasts or hemocytes There are bad knife marks going up and to the right The dark spots at the bottom right are not A viridans because this did not come from the infected lobsters I guess that the black spots are granules There is a tiny bit of focus texture but considering this is an extremely low-mag image that is a lot of focus texture compared to the high-mag images But that is ok because this image is so low mag that the focustexture is barely visible over the pixilation so it does not obscure anything There is good stigmation

33

34

Fig 14 Thomas Lipscomb 110 Grid E3 LDG endoplasmic reticulum

This micrograph was chosen because it is the one with the most cellular organelles visible all across There is rough endoplasmic reticulum in the middle and smooth endoplasmic reticulum at the bottom and upper right (smooth ER tends to have more circle-shaped parts in the section than rough ER) This is the only example of smooth ER in the report It may even be possible that the curved membranes in the center of the image are golgi because golgi tends to be curved There are about two dark circles visible one on the upper left right above the white circle and one on the right However cristae are not visible so it is not certain that they are mitochondria The focus texture is pretty mild and one of the best of all the micrographs The white circles are too light and uniform to be nuclei Perhaps they are lipid storage vacuoles But the white circle on the left is where I would expect a nucleus to be (nestled in the rough ER) Maybe the plane ofsection missed the nucleus There are no knife marks but the upper left is darker probably because of uneven illumination There is good stigmation

35

36

Fig 15 Thomas Lipscomb 111 Grid E3 LDG rough endoplasmic reticulum zoomed 110

This is rough endoplasmic reticulum The ribosomes are very clear dots along the membranes There is moderate focus texture especially considering the mag is only 36000x But somehow the image transcends the bad focus texture because the dots of the ribosomes are somehow much clearer than expected with the bad focus texture The membranes are curved so this could possibly have been golgi except that it has ribosomes attached to it The illumination is uneven the lower left is much darker than the upper right It is either knife marks causing uneven thickness of section or an off-center beam of electrons We already know that Grid E3 has moderate to bad knife marks There is good stigmation

37

38

Fig 16 Thomas Lipscomb 116 Grid E3 LDG lipid storage vacuoles and microvilli on lower left

The LDG like the human liver stores lipids in lipid storage vacuoles The lipid storage vacuolesare the light-colored spheres The microvilli of the LDG are in the lower left so these lipid storage vacuoles are pretty close to the lipidsrsquo point of origin inside of the food in the digestive tubules of the LDG Cells like these tend to have the nucleus and ER as far away from the microvilli as possible (Factor 2014) so they are safe and do not get in the way I assume that these lipid storage vacuoles are travelling from the microvilli in the lower left towards the blood space past the upper right of the image to transport the food from the LDG to the rest of the lobster There are no nuclei ER golgi or mitochondria in this micrograph The dark spots at the top left and bottom are not A viridans because this did not come from the infected lobsters I guess that the black spots are contamination because they do not have the regularity in size and shape that granules have There are bad knife marks (going up and to the right) in this image There is mild focus texture but considering that this is 4400x that is actually moderate focus texture There is good stigmation

39

40

Fig 17 Thomas Lipscomb 118 Grid E3 LDG nucleus and granules

This image has good detail of the nucleus (in the center) and some granules (at the bottom) The cell membrane appears to be in the bottom left No mitochondria or ER are visible There is clearly some heterochromatin (dark stain) in the nucleus and the lighter stains are euchromatin The knife marks are moderate and vertically aligned There is not much focus texture and there is good detail The dark spots at the bottom are not A viridans because this did not come from the infected lobsters the dark spots are probably granules but there are not enough of them for me to call this cell a granulocyte There is good stigmation

41

42

Fig 18 Thomas Lipscomb 121 Grid E3 LDG fixed phagocyte

This is a fixed phagocyte with a strange focus effect that Dr Factor has seen before and does not understand (Factor 2014) The ridges of the fixed phagocyte (which increase surface area) can be seen in the cell membrane and make it look like a cauliflower The nucleus is in the middle and strangely there are no clear areas dominated by heterochromatin (dark stain) There might be a nucleolus in the upper left of the nucleus Some of the spheres that are not as dark as the granules may be mitochondria but cristae cannot be clearly seen at this magnification Extreme knife marks going from top to bottom are seen There is moderate focus texture and this is low-mag so that is a lot of focus texture The ER and golgi are not visible in this section There is good stigmation

43

44

Fig 19 Thomas Lipscomb 124 Grid E3 LDG microvilli

This is the only high mag image of the microvilli in the LDG in this report The microvilli are at the top of the image and are a forest of tubes that increase the surface area of the cell membrane to increase the rate of food absorption (Factor 2014) Figures 11-18 had less contrast than what Ithought was optimal so this figure was made using objective aperture 3 (30 microm) of the TEM notthe standard objective aperture 2 (50 microm) that were used for Figures 1-18 At the bottom of this image is what appears to be a lipid storage vacuole I was surprised at the lack of detail and structures in the cytoplasm between the microvilli and the lipid storage vacuole The nucleus and its associated endoplasmic reticulum and golgi would not be in this portion because it would be at the basal end of the cell not the apical end with the microvilli (Factor 2014) The mitochondria might be at this apical end but none are visible in this section There are bad knife marks going up and to the left which we have seen before in grid E3 There is moderate focus texture and this is a moderate-mag image There is good stigmation

45

46


Figures 11-18 had less contrast than what I thought was optimal so this figure was made using objective aperture 3 (30 microm) of the TEM not the standard objective aperture 2 (50 microm) that wereused for Figures 1-18 In the middle of this image is the nuclear membrane The nucleus is in the upper right corner (not the upper left as the image caption says that was a mistake) The cytoplasm is in the lower left corner The nuclear membrane in-between can be seen clearly but unlike the other high-mag micrographs the dark lines are not very visible There is very bad focus texture the worst in the entire report Maybe the dark parts are not as clear because the LDG was not stained as heavily as the gill Without strong dark parts to focus on I had much more trouble focusing this image because I could only do it based on the worminess of the background which may be why the focus is so off There is good stigmation

47

48

Discussion

Of course both Gill and LDG have nuclei rough and smooth endoplasmic reticulum golgi and mitochondria However smooth ER was only found in LDG (Figure 14) and that makes sense because there should be a lot more smooth ER in LDG because there is a lot more for smooth ER to do in LDG (handling lipid storage vacuoles for example) than in gill Golgi was not conclusively found anywhere because I am not good enough at identifying it I only look for unusually curved endoplasmic reticulum to say it might be golgi

I got good images of blood spaces in the gill (Figure 2) and the LDG (Figure 13) Granulocytes were found in both Gill (Figure 4) and LDG (Figure 12) A fixed phagocyte was only found in the LDG (Figure 18) not the gill even though both should have them (the fixed phagocytes filter the blood) Lipid storage vacuoles were found in the LDG (Figures 12 13 14 and 16) not the gill as expected Microvilli were found in the LDG (Figures 16 and 19) not the gill as expected The gill also have microvilli-like structures radiating from the circular vessel (Factor 2014) but they are much larger than microvilli because they need the space to have theirown blood circulation Aerococcus viridans were only found in the gill not the LDG as expectedbecause only the lobster that the gill came from was infected with A viridans not the lobster thatthe LDG came from

49

Literature Cited

1 Factor JR ldquoTransmission Electron Microscopy and Cell Ultrastructurerdquo BIO 4650 classlecture SUNY Purchase Purchase August 25 2014 to December 11 2014 PowerPoint lecture

2 Factor JR 1995 Biology of the Lobster Homarus americanus (First Edition) WalthamAcademic Press Inc Chapter 15 The Digestive System pp 409-421

3 Appendix 3 TEM Plastic Araldite 502Embed-812TM Embedding Tracking Form TEM Embedding Procedure ndash For Araldite 502Embed-812 TM Plastic (Procedure for Tissues without Polystyrene Microspheres)

4 Surace F 2012 Testing for Capsules in Aerococcus viridans The Disease Agent for Gaffkemia in American Lobsters SUNY Purchase Biology BA thesis

5 Factor JR 2012 Operating Instructions for FEIPhilips Morgagni (M268) Transmission Electron Microscope

6 Granulocyte - Wikipedia the Free Encyclopedia (2014 November 16) Retrieved December 14 2014 from httpenwikipediaorgwikiGranulocyte

50

Introduction

This series of transmission electron microscope micrographs images two organs in Homarus americanus lobster digestive gland and Aerococcus viridans-infected gill Aerococcusviridans-infected gill is being investigated because the lab is trying to understand the pathology of Aerococcus viridans especially how they survive uptake into the lobster by vacuoles and then how the filaments that Aerococcus viridans releases might be the cause of the vacuoles pathologically growing too large Non-infected lobster digestive gland is being investigated by me because it is the control group Aerococcus viridans-infected lobster digestive gland is an experimental group and others are comparing it to the non-infected lobster digestive gland healthy tissue

I hope to see the gill tissue (and even the blood spaces feeding it) containing pathologically large vacuoles containing Aerococcus viridans I hope to see the cuticle of the gill granulocytes mitochondria smooth and rough endoplasmic reticulum and nuclei I hope tosee the lobster digestive gland containing microvilli fixed phagocytes more granulocytes more endoplasmic reticulum more nuclei and more mitochondria

One point of comparison between the tissues of the gill and LDG is that gill is an Aerococcus viridans-infected tissue and the LDG is not The blood spaces in the gill and the blood spaces in the LDG can be compared The gill filaments are similar in structure to the LDGrsquos microvilli but the gill filaments are much larger The cell components of the cells in the gill and the cells in the LDG can be compared endoplasmic reticulum mitochondria nucleus and vesicles I did not find golgi anywhere even though it should be present (Factor 2014)

The lobster digestive gland is located in the midgut (Factor 1995) It is primarily responsible for the synthesis and secretion of digestive enzymes the final digestion of food and absorption of nutrients (Factor 1995) It is also involved in excretion lipid and carbohydrate metabolism storage of lipids and storage of inorganic reserves (Factor 1995) The lobster digestive gland is a pair of large complex glands each with its own duct lying primarily in the cephalothorax (Factor 1995) Each gland is divided into three lobes (Factor 1995) Each lobe has a series of blindly ending digestive tubules that connect to the duct (Factor 1995) The tubules are approximately 100 microm in diameter (Factor 1995) Circular and longitudinal contractile bundles of myofilaments lie between the basement membrane and the tubules (Factor1995) Microvilli increase the surface area of the LDG tubules (Factor 2014) The tubules are interspersed with hepatic arterioles that are covered in fixed phagocytes (Factor 1995) The arterioles supply the tubules with blood (Factor 1995) The fixed phagocytes contain electron-dense granules and are ridged to increase surface area (Factor 1995) The fixed phagocytes are part of the lobsterrsquos immune system by filtering foreign material from the bloodstream and beinga barrier between any germs that might enter from the ingested food in the LDG into the bloodstream (Factor 1995) The fixed phagocytes engulf germs like Aerococcus viridans but Aerococcus viridans somehow survives in the phagocytic vacuoles and over time the phagocytic vacuoles become pathologically large perhaps because of the filaments that Aerococcus viridans secretes (Factor 2014)

The lobster gill contains two blood vessels the branchial stem afferent vessel that carries blood from the gill to the heart and the branchial stem efferent vessel that carries blood away from the heart to the gill (Factor 2014) Those two blood vessels are next to each other and

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This is a survey image of LGD that tries to capture the morphology of the entire tissue in one image with the epithelium and the basal lamina below it being the horizontal line at the top and the rest of the gill below it 11 of the book Biology of the Lobster Homarys americanus was used to label the parts of the tissue in this micrograph (Factor 1995) Just beneath the epithelium should be a band of dark-colored circular muscle The large light-colored parts belowthat are probably blood sinuses The dark colored portions below the circular muscle could be basophilic granulocytes fibroblasts or hemocytes There are bad knife marks going up and to the right The dark spots at the bottom right are not A viridans because this did not come from the infected lobsters I guess that the black spots are granules There is a tiny bit of focus texture but considering this is an extremely low-mag image that is a lot of focus texture compared to the high-mag images But that is ok because this image is so low mag that the focustexture is barely visible over the pixilation so it does not obscure anything There is good stigmation

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lipscomb, thomas - tem'14 - report

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