1) Cells and microscopy Flashcards
What is the typical magnification of a light microscope?
up to 1500x
What is the typical resolution of a light microscope?
limit of 200nm
How can resolution be improved?
use radiation with a shorter wavelength
Define magnification
the number of times larger an image appears compared to the real specimen
Total magnification = ?
eye piece magnification x objective magnification
Magnification = ?
size of image / size of object
Define resolution
the ability to distinguish between two distinct objects separately and to see detail
1mm = _m?
1 x 10^-3
1μm = _m?
1 x 10^-6
1nm = _m?
1 x 10^-9
Why are stains used?
to make different parts of the specimen stand out more clearly as often colourless and almost transparent
What colour does iodine solution turn starch grains?
blue-black
Name two stains which can be applied to living cells directly
methylene blue
iodine solution
Some specimens need to be fixed with _ or _ to make proteins and nucleic acids insoluble (killing cells)
acetic acid
alcohol
Give 4 features of cell theory
- the cell is the basic unit of all life forms (uni/multicellular) and the smallest unit capable of surviving independently
- metabolic processes take place inside cells
- new cells are derived from old cells
- cells possess genetic material passed on from parent to daughter cells
Give the journey of light through a light microscope
bulb under stage -> condenser lens -> specimen -> objective lens -> eyepiece lens
Why does a light microscope contains one or more lenses?
to focus light into the eye
Give 3 advantages of a light microscope
- observation of a wide range of specimens (inc. living), smear preparations and thin sections of tissue
- easy to use (no specialist training required) and transport
- relatively inexpensive
Give 1 disadvantage of a light microscope
limited resolution so most internal cellular structures are not visible
Define electron microscope (EM)
microscope that uses a beam of e- as its source of radiation to view a magnified image of an object
An electron microscope has a greater _ than a light microscope
resolution
Describe how an electron microscope works (3 points)
- electrons focused onto a fluorescent screen which emits visible light where electrons hit
- forms a photomicrograph (visible image on screen)
- final images are black, grey and white however, colour can be added by specialist computer programs to produce false-colour electron micrographs
Give 3 features of a transmission electron microscope (TEM)
- thinner specimens (e- less penetrating)
- heavy metals are used to stain specimens (large +ive nuclei scatter e- so don’t hit fluorescent screen and leave a dark area) so structures that take up stain appear as dark images
- 2D, black + white image
Give 4 features of a scanning electron microscope (SEM)
- e- don’t pass through specimen, instead are reflected off its surface
- beam of e- passed back and forth over surface of specimen in regular pattern
- pattern of reflected e- / scattered e- reflects contours of specimen
- 3D image produced with computer analysis
Why would a confocal microscope be used?
to obtain high resolution images and 3D reconstructions
to produce focused images of thick specimens at various depths using optical sectioning
What can fluorescent markers be used for?
the detection of biological objects
Name 5 key parts of an electron microscope
electron source magnetic condenser magnetic objective magnetic projector fluorescent screen
Give 1 advantage of an electron microscope
greater resolving power (0.1nm) with a higher magnification means smaller objects can be seen in more detail
Give 5 disadvantages of an electron microscope
- large + expensive
- requires trained personnel and specially designed room
- specimens in near vacuum environment (air molecules absorb e-) so must be dehydrated + dead
- complex staining process
- artefacts may occur due to preparation techniques
What does using a preserved form of specimen enable?
the specimen can be cut into sections and treated with a variety of stains to reveal different structures within the tissues and cells
Name the 3 stages temporary slide preparation
fixation
staining
mounting
What 2 key things does temporary slide preparation require? Why?
70% alcohol and thin glass coverslip
to exclude dust and air; to protect high power objective lens of microscope
Name the 3 stages permanent slide preparation
fixation dehydration clearing embedding sectioning staining mounting
What will a specimen be mounted in if the specimen is likely to dry out or a prolonged examination is likely?
glycerine
Which type of blood cell can clearly be seen under a microscope?
erythrocytes (red blood cells)
Why do white blood cells require staining?
to distinguish between the different types by showing differences in the shapes of their nuclei
What do different structures vary in?
the type / quantity of stain they take up
What is the benefit of the staining process being automated in a laboratory?
allows many slides to be prepared at the same time
Why may a blood smear be prepared and examined?
to observe the appearance of blood for diagnostic tests (identify cells and pathogens present)
Name 3 types of section
longitudinal
transverse
oblique
Name 3 differential stains for plants, their final colours and what type of tissue each are suitable for
eosin -> red = cellulose
toluidine -> blue = lignin
or -> purple = cellulose
iodine KI -> blue-black = starch
Why is toluidine used for plant vascular tissue?
stains lignified cell walls blue and cellulose cell walls purple
to allow xylem vessels to be distinguished
Name 2 Romanowsky stains for blood
Leishman’s stain
Wright’s stain
Give the 5 steps for using Leishman’s stain
- blood allowed to dry
- fixation with methanol
- flooded and left for 2 mins
- diluted with water and left for 5-7 mins
- washed until pale pink to the naked eye
When is Wright’s stain used?
for differential white blood cell counts when infections suspected
Give a brief 1 sentence description of how a blood film / smear is made
a sample of blood is spread onto a microscope slide (usually by machine)
What happens if a blood smear is too thick?
individual cells cannot be seen
What happens if a blood smear is too thin?
small number of cells may not be representative
Give the 5 steps for preparing a blood smear by hand
- small drop of blood at the end of a sterile, clean and dry microscope slide
- spreader (sterile slide) pushed along slide at a 30 degree angle to spread blood
- label with patient’s details
- dry in air (cells stick to slide)
- fixative to apply cells
What are the 4 key components of the blood?
plasma
red blood cells
white blood cells
platelets
What are the 6 components of blood plasma?
water proteins glucose mineral ions hormones carbon dioxide
What are the 7 key functions of the blood?
- delivery of oxygen and nutrients to tissues
- removal of waste from tissues
- immunological protection
- clotting
- transport of cell signalling molecules (e.g. hormones)
- acting as a buffer to regulate body pH (acidity)
- distribution of heat to regulate core body temperature
How should biological drawings be drawn? (4)
in pencil
single, clear continuous line
labels and annotations
total magnification used
What are the 2 types of leucocytes?
granulocytes and agranulocytes
What type of blood cell is an example of a granulocyte?
neutrophil
What 3 types of blood cell are examples of agranulocytes?
monocytes
lymphocytes
macrophages
What is the function of erythrocytes?
deliver oxygen from lungs to the body and carbon dioxide from tissues to lungs
Where are erythrocytes developed?
in the bone marrow
How are erythrocytes developed?
from erythropoietic stem cells by erythropoiesis (stimulated by erythropoietin, EPO)
How long does the development of erythrocytes take?
7 days
What are immature erythrocytes called?
reticulocytes
How can red blood cells be identified?
methylene blue stain
How long do erythrocytes circulate for?
100 - 120 days
Give 3 key features of erythrocytes
- contain haemoglobin (binds reversibly with oxygen to form oxyhaemoglobin)
lacks nucleus and most organelles so… - large surface area to volume ratio
- biconcave (flexible to squeeze through capillaries)
What is the diameter of an erythrocyte?
6.2 - 8.2 μm
What is the width of an erythrocyte at its thickest point?
2 - 2.5 μm
What two features of an erythrocyte should be included on its biological drawing?
cell surface membrane
cytoplasm (stained darker at edges and lighter in centre)
What is an alternative name for platelets?
thrombocytes
What is the function of platelets?
contribute to blood clotting and clot formation
Where do platelets develop?
in bone marrow
How long to platelets circulate for?
6 - 7 days
Give 3 features of platelets
biconcave discs of cytoplasm fragments surrounded by a cell surface membrane
no nucleus
appear as dark purple spots when stained
What is the diameter of a platelet?
2 - 3 μm
What is the function of leucocytes?
immunological protection
Describe granulocytes
contain granules (often lysosomes) in their cytoplasm which stain differently
Describe agranulocytes
absence of granules in cytoplasm
Leucocytes are _ in size than erythrocytes but _ abundant
larger
less
What is the function of neutrophils?
defend against fungal / bacterial infection by phagocytosis (engulfing / breaking down bacteria)
How long do neutrophils circulate for? Why?
5 days
unable to renew lysosomes so die after breaking down a few pathogens
Give 1 key feature of a neutrophil
multi-lobed nucleus - increases flexibility to be able to leave capillaries by squeezing through fenestrations in the capillary wall
What 3 features of an neutrophil should be included on its biological drawing?
cytoplasm - stained light blue
cell surface membrane
multi-lobed nucleus - stained deep blue; no. of lobes vary between 2 + 5
What are lymphocytes characterised by?
large, deeply stained nucleus surrounded by relatively small cytoplasm
What are the 2 types of lymphocyte?
B and T
What is the function of B lymphocytes
produce immunoglobins (antibodies)
Name the 3 types of T lymphocytes
helper T cells
cytotoxic T cells
natural killer cells
What is the function of helper T cells?
produce cytokines and co-ordinate immune response
What is the function of cytotoxic T cells?
bind to antigens on virus infected / tumour cells and destroy them
What 3 features of an neutrophil should be included on its biological drawing?
cell surface membrane
nucleus - circular; stained dark purple; large
cytoplasm - small quantity; pale purple
What is the function of monocytes?
phagocytosis
leave bloodstream and differentiate into tissue macrophages (remove cell debris and attack microorganisms) found in lungs, lymph nodes and liver
Monocytes circulate for a _ time than neutrophils because _?
longer
they are able to replace their lysosomes
What 3 features of an neutrophil should be included on its biological drawing?
cell surface membrane
nucleus - darkly stained; kidney bean shaped
cytoplasm - stained purple
