Analysis of Blood with a Light Microscope

by Dr. Robert Berdan

April 27th, 2021

Fig. 1. Above is a picture of a human blood smear consisting of two white blood cells surrounded by red
blood cells and a few platelets after staining with Wright’s stain. 1000X oil immersion objective bright
field microscopy. The arrow points to a Barr body which is a small drumstick found attached to the
nucleus that represents an “extra” inactivated X chromosome indicating this blood sample is from a
female.

Introduction

Blood analysis is an important diagnostic tool and can be even be used to determine the sex of an
individual. One theory suggests that the reason we may be attracted to the colour red more than any
other is because it might alert us to injury i.e. we are bleeding. When we cut ourselves we bleed and if
the cut is small it usually clots within minutes due to blood platelets. The most numerous blood cells are
red blood cells (erythrocytes) that carry oxygen from our lungs to the various tissues in our body. Plasma
is a clear-yellow fluid that also contains several different types of white blood cells and each type of cell
plays a specific role in our immune defense. Blood plasma also contains nutrients, amino acids, fatty
acids, sugars and hormones and may contain pathogens.

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 If you are unwell your doctor may send you to get your blood tested if it is suspected that you might
have an infection, disease or other ailment. What the blood is tested for depends on what you doctor
suspects might be wrong. Testing could include chemical analysis, or analysis of the different blood cell
types or both. In Canada laboratory personnel that do the testing require several years of training. If
your doctor suspects cancer or some other ailment the blood samples will also be inspected by a
histopathologist whose training requires a bachelor’s degree, a degree from Medical school and 3-7
years internship and residency program. I mention this because there is another group that examines
live blood with a light microscope and their training program lasts only a few weeks. This Live blood
analysis group looks at your blood using dark field microscopy. This group cannot diagnose illnesses but
will show you your live blood with a microscope and suggest changes in your diet or adding supplements
to improve your health. They should not and cannot diagnose illness – see Live Blood Analysis
(Wikipedia). For the most part analysis of blood is quantitative, the cells are stained (.e.g. Wright’s stain)
and the number of platelets, and different types of white blood cells are counted manually or
automatically with a flow cytometer. In addition the shape and size of red blood cells are analyzed. In
some instances bacteria, fungi and parasites can be detected in blood smears. Proper blood analysis
involves a team of trained experts, though anyone with a light microscope can examine blood of humans
or animals.

Blood analysis for both humans and animals is usually conducted by staining the cells and observing
them by bright field microscopy. Other microscopy techniques like phase contrast; differential inference
contrast (DIC), dark field microscopy, and immunofluorescence can also be used. In this introductory
article I show different white blood cells examined by a variety of microscopy techniques.

Staining Blood

Bright field microscopy is used to view blood cells after making a blood smear as shown in Figure 2
and after staining with Wright’s stain (there are other stains that can be used as well). Place a small
blood spot on one side of a glass microscope slide after pricking a finger, use a second slide to contact
the blood spot which spreads across the top slide edge by capillary action. Then pull the top slide at
about a 30 degree angle to form a thin blood smear. Allow the blood to dry for a few minutes and then
stain the cells. For staining I put the slides in Petri dishes and flood the surface of the blood smear with
Wrights stain for 2- 5 minutes, then add buffer solution for 2-5 minutes, rinse the slide in buffer and dry
the slide in air. I cover the slide with Permount™ (Fisher Chemical) and a 50 mm long coverslip and view
the slide using bright field microscopy. It takes about 15 minutes to make a prepared blood smear. With
practice good slides provide an even distribution of blood. I may heat the slide for a few moments on a
hot plate set on low so the Permount™ spreads and the coverslip dries flat. To see details in the white
blood cells and identify (platelets) also called thrombocytes use a 40X, 60X or 100X objectives. The 100X
objective with oil immersion fluid is required to get clear views of the white cells and see details in the
nucleus.

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Fig. 2. Above shows the steps in how to make a blood smear. 1. Put a drop of blood on a microscope
slide 2) Pull another slide to the right so it touches the blood drop and the blood moves by capillary
action along the edge of the slide then 3) move the slide at approximately at a 30 degree angle away
from the drop of blood to make a thin smear, dry the slide and stain the blood cells. Wrights stain from
Benzmicroscope.com.

The red blood cells are about 7-10 microns in diameter; they are biconcave disk shaped and have no
nuclei. You might see some immature red blood cells with some dark blue staining spots that represent
ribosomes and messenger RNA. The shapes of the red blood cells may change slightly depending on the
external solution, or whether the person has a disease e.g. sickle cell anemia. Red blood cells may stack
in what is called a Rouleaux formation which can often occur at the edge of the blood smear, or it can be
due to infection, multiple myeloma, inflammatory conditions, pH changes, diabetes and cancers.
Fibrinogen may interact with sialic acid on the surface of the red blood cells to facilitate the formation of
rouleaux (Wikipedia). Generally examine the slide in the central regions not at the edges. Also beware
that viruses can not generally be detected by an ordinary light microscope, unless viruses are stained
with fluorescent antibodies after which they may appear as bright coloured dots.

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Fig. 3 Human white blood cells after staining with Acridine orange and viewed by fluorescence
microscopy. A) Neutrophil B) Lymphocyte c) Eosinophil? Acridine orange stains DNA green, and mRNA
red. Scale bar = 10 microns (0.010 mm). 1000X

Fig. 4 Live blood sample viewed by Phase contrast microscopy 1000X. The majority of the cells in this
picture are red blood cells. Near the top of the picture are two white blood cells next to each other, but
without staining they can’t be definitively identified. With phase contrast the biconcave shape of the red
cells creates the appearance of concentric rings in the red blood cells (erythrocytes). Phase contrast
microscopy is useful for detecting some bacteria or other pathogens.

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Fig. 5 A scanning electron microscope (SEM) image of a normal red blood cell (left), a platelet (middle),
and (right) a white blood cell courtesy National Cancer Institute
https://en.wikipedia.org/wiki/Blood#/media/File:Red_White_Blood_cells.jpg B Neutrophil engulfing
bacteria Bacillus anthracis. Photo by Volker Brinkmann - (November 2005) PLoS Pathogens 1 (3): Cover
page. DOI:10.1371. Scale bar = 5 microns. Images are false coloured as SEM photos are generally black
and white.

White Blood cells

In a Wright’s stained blood smear white blood cells have distinct shaped nuclei and can be easily
distinguished. White blood cells play important roles in the body’s immune response. Changes in the
number of specific white blood cells can indicate an allergy, or disease. Individuals infected with COVID-
19 virus often have decreased numbers of Lymphocytes (T, B, NK cells) and the overall white cell counts
are elevated, especially the neutrophils (Han, H. et. al. 2020). COVID-19 patients also exhibited elevated
monocytes and coagulation abnormalities which can cause blockage of blood vessels (L. Thomas, 2021
and M. Kubankova et al. 2021).

The diagnosis of any kind of disease should include a clinical history, physical examination and
laboratory testing. The presence of bacteria in blood (septicemia) or parasites is serious and demands
immediate attention by a doctor. Also see “The study of blood and its contents” a web site with more
pictures of blood cells and various ailments (J. Sprute).

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Fig. 6 Different white blood cells stained with Wright’s stain – A Neutrophil, B Neutrophil with a Barr
body C lymphocyte D Eosinophil E Monocyte F Basophil G Stab cell – young Neutrophil H platelets
(thrombocytes ). Bright field microscopy 1000X.

Macrophages and Monocytes

Monocytes are the largest white blood cell and can differentiate into macrophages and dendritic
cells. They are amoeboid in appearance and lack granules in their cytoplasm. These large cells can be
about 20 microns in diameter with a large and/or bean shaped nucleus. The nucleus is not divided into
lobes and the cells are present from 2-10% of the white blood cells. Monocytes are attracted to
damaged tissue and are induced to become macrophages which engulf tissue and debris. These cells can
persist for several months. In the lungs of a person exposed to asbestos macrophages try to engulf the
asbestos particles which may damage the macrophage’s nucleus and in turn some of these cells can
become cancerous.

Neutrophils

Neutrophils are the most abundant type of white cells consisting of 40-70% of white cell population.
These short lived cells (live 5-35 hours) are easy to identify due to their segmented nuclei in stained
blood preparations. Neutrophils can have from 2-5 nuclei, however they can exhibit hyper segmentation
(6 or more nuclear segments) if there is a Vitamin B deficiency. Neutrophils with a single nuclear band
are called stab cells and they are immature cells. When the cells are activated they change from a
circular to more amoeboid shape and engulf bacteria and other pathogens. White blood cells from
females may exhibit a small drum stick shape structure attached by a fine thread to the nucleus in up to
17% of cells (W.M. Davidson and D.R. Smith (1954). These are inactive X chromosomes and females
have two X chromosomes one is inactivated. These inactive X chromosomes are called Barr bodies

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named after the discovery by Dr. Murray Bar and his graduate student E. G. Bertram in 1949 at the
University of Western in London, Ontario.

Eosinophils

Eosinophils are white blood cells 12-17 microns in size that play a role in allergic responses and
asthma. They consist of usually less than 7% of white blood cells. Their cytoplasmic granules stain with
acidophilic dyes like eosin that appear pink. The eosinophils stay in circulation for about 8-12 hours.
Eosinophils move to inflammatory sites where they are activated and release their toxic granules. Some
eosinophils fight viral infections and worm colonization. They have a band shaped nucleus surrounded
by pink granules.

Basophils

Basophils are a type of white blood cell with large granules that obscure the nucleus when stained.
When unstained their nucleus has two lobes. They make up 0.5 to 1% of white blood cells and play a
role in the immune response and inflammation. They are involved in asthma, atopic dermatitis (eczema)
and hay fever. They are called Basophils because they stain with basic dyes. These cells contain
histamine which is a vasodilator and heparin that prevents clotting.

Platelets (Thrombocytes)

Platelets are cytoplasmic fragments derived from multinucleated giant cells called Megakaryocytes of
bone marrow. In blood stained with a basophilic stain they appear as blue clusters (see below). They
appear as oblate spheroids 2-3 microns in diameter and range from 200,000 to 450,000 per microliter.
They are only found in mammals, in birds and amphibians they circulate as intact mononuclear cells.
They can also be activated by abnormalities in blood vessel walls resulting in inappropriate clots
(thrombosis) obstructing blood flow. They can be measured and counted with a microscope and
hemocytometer slide.

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Fig. 7. Erythrocytes (red blood cells) and platelets (blue) – Blood smear Wright’s stain 1000X Oil
immersion bright field microscopy.

Barr Bodies or Sex Chromatin

M.L. Barr and E.G. Bertam (1949) were the first to notice dark chromatin staining structures
in some cat nuclei and noted they were only found in female cats. Later on W.M. Davidson and
Dr. Smith (1954) noticed drum stick-like structures attached to the nuclear lobes of some
female neutrophils. Today we know that these structures are inactive X chromosomes. Females
have 2 X chromosomes and one inactive X chromosome can be detected by staining. Several
studies have confirmed they are present in a percentage of neutrophils. Barr bodies can also be
visible in some men with Kleinfelter’s syndrome with a 47XXY chromosomal karyotype. Barr
bodies can be also seen in cheek epithelia cells and some hair cells after staining. The Barr
bodies are about 1 micron in size.

Lyme disease

Lyme disease is caused by a bacteria spread by deer ticks to humans and if not diagnosed can cause
serious symptoms. The bacteria can sometimes be detected in blood smears as shown below.

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Fig 8 Above is a photo of a blood smear showing Borrelia burgdorferi, a spirocheate bacteria (appear as
wavy lines) 1000X Oil immersion with bright field microscopy. This bacteria from deer ticks causes Lyme
disease.

Lyme disease is caused by bacteria Borrelia burgdorferi (C. Kurokawa, 2020). They are difficult to
detect immunologically or in blood smears by light microscopy. Lyme disease is spread by ticks (Ixodes
scapularis – a deer tick) and anyone hiking outdoors should be aware of the ticks. I have lead
photography tours in the Canadian Rockies and mention them to my groups especially in spring. For
photographers simply putting their camera bag on the ground can attract ticks where they attach
themselves and later crawl up into the persons hair, then when asleep they crawl onto your body suck
your blood and infect you. You likely won’t feel the tick bite, but the ticks engorge themselves with
blood and can infect you. When I was bitten by a tick my doctor put me immediately on the antibiotic
Erythromycin for 10 days and I was OK (I don’t know if the tick was infected). Some of my friends have
not fared as well.

First thing after a day hiking in regions where the ticks are common e.g. around deer, big horn sheep,
small mammals etc, you should take a shower and wash your hair to get rid of any ticks before going to
bed. If you can’t shower check your hair and body for ticks before going to sleep. Some folks exhibit an
expanding red rash about a week after being bitten. If you get a rash visit your doctor as soon as
possible and alert the doctor where you were hiking. The rash is not itchy or painful and only develops
in about 75% of people. Early symptoms of Lyme disease may include fever, headache and fatigue. The
disease is not transmittable between people, animals or via food. If you exhibit tick bite symptoms and
have been walking in an area where ticks are found let your doctor know. Immunological tests can be

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negative so get a 2nd test a few weeks later or take the prescribed antibiotics. In 10-15% of untreated
people Lyme disease can cause neurological conditions which can occur 1-12 weeks after a tick bite.

Live Blood Cell Analysis

Live Blood Cell Analysis uses dark field light microscopy to observe a fresh blood sample. This analysis
is not accepted in standard laboratory practice but is considered a form of alternative medicine. Live
Blood Analysis is not regulated and anyone with a light microscope with dark field accessories can
become a practitioner. Training involves a four week course, though some practitioners may have
additional training in nutrition. Live blood cell practitioners cannot legally diagnose or treat medical
illness. They offer live views of your blood on a computer monitor and may suggest nutritional
supplements. Some practitioners have been incriminated and charged for making incorrect claims
(Wikipedia). As a scientist I can appreciate the attraction of seeing your own blood, though caution
anyone to beware of Live Blood Cell Analysis claims and see your doctor if you have any concerns or
wish to verify any diagnoses you may be given on the basis of live blood analysis.

If considering live blood analysis do some Internet research (e.g. Read letter by Tiffany Clouston who
teaches Blood Technologists in Canada), Wikipedia and other credible medical web sites -
https://www.csmls.org/Advocacy/Public-Awareness/Smoke-and-Mirrors-An-Inside-Look-at-a-Live-
Blood).

Fig. 9. Above photo shows live blood cells by Dark field microscopy 100X. Most of the cells shown are
red blood cells. High magnification dark field microscopy can detect bacteria, some pathogens and
alterations in blood cells.

Note that whereas many standard light microscopes can produce dark field at lower magnification

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(10X, 20X and 40X objectives) using special opaque disks, or the “wrong” phase condenser disk, at
higher magnification (60X or 100X objectives) dark field microscopy requires a special dark field
condenser with a numerical aperture greater than that of a high power objective and uses oil immersion
fluid between the condenser and bottom of the glass slide in addition to the coverslip and objective
(Bagnell 1964). A Dark field 100X objective may also have an iris diaphragm built in to fine tune dark
field light (C. R. Bagnell, 2012).

If you have a serious illness that requires blood analysis I urge you to visit your medical doctor and a
get traditional blood analysis. The costs of traditional blood tests are paid for in Canada, while those of
Live Blood Analysis are not. If you want to see your blood and get nutritional advice then live blood
analysis is an option. An alternative option if you want to observe blood is to consider owning your own
microscope.

Fig. 10 Live blood cells using Differential Interference Contrast microscopy 1000X. The arrows point to
two white blood cells and the asterisk (*) shows some crenated red blood cells which can occur in live
blood samples or blood cells bathed in a hypertonic solution. Scale bar = 10 microns.

Animal Blood Cell Analysis

Veterinarians conduct animal blood analysis for a variety of reasons. Blood from other mammals
appears microscopically similar to human blood. Blood cells from birds, reptiles, fish etc., look different
being oval and have red blood cells with nuclei. Being able to analyze and distinguish animal blood from
human blood is important in Forensic science. One research study used morphometric analysis
(measured the diameter and circumference) of red blood cells to distinguish blood from cattle, sheep,
goats, horses and dogs using both a light microscope and software (N. Adili et al. 2016). There are

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Canadian animal blood banks for injured animals (http://www.canadiananimalbloodbank.ca/). A new
technique to differentiate human and animal blood uses total reflection Fourier transform-infrared
spectroscopy (E. Mistek-Morabito and I. K. Lednev (2020). Pet owners can have their animal’s blood and
urine tested by a Veterinarian.

Fig. 11. Frog red blood cells from a prepared microscope slide. The red blood cells have a nucleus -
1000X

Fig. 12. Fish red blood cells have nuclei - photograph of cells on a prepared slide 1000X.

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Summary & Conclusions

Blood cells from mammals appears similar microscopically to cells in humans, whereas red blood cells
from birds, frogs and fish have nuclei. In humans blood contains several different types of white blood
cells each with specialized functions and the number of specific white cells is diagnostic as is the shape
and number of the red blood cells. Blood analysis by microscopy can be used to detect parasites,
bacteria, fungi and other pathogens. Live blood analysis utilizes dark field microscopy to investigate
blood, is controversial and practitioners are not qualified to make medical diagnoses. Anyone with a
microscope can view blood cells, but caution is required in handling other people’s blood because it
might carry infectious agents (HIV human immunodeficiency virus, COVID-19, Lyme disease etc.). One
should wear rubber gloves when handling human blood. Motic offers a wide variety of light
microscopes suitable for blood analysis and a sales person can advise you on which microscope is most
suitable for your needs and budget if considering purchasing a microscope..

Acknowledgements

I would like to thank several people for suggestions on this short essay: Cindy Larsen (blood
technologist), Mike Codner (amateur microscopist), Brandon Berdan and Dr. Sharif Galal MD.

References

L. Thomas (2021) What are the stages in the physical phenotype of blood cells in COVID-19? News
Medical Life Sciences. https://www.news-medical.net/news/20210216/What-are-the-changes-in-the-
physical-phenotype-of-blood-cells-in-COVID-19.aspx#

A. Kubánková et al. (2021) Physical phenotype of blood cells is altered in COVID 19 (Preprint). BioRix
https://www.biorxiv.org/content/10.1101/2021.02.12.429482v1

H. Han et al. (2020) Descriptive, Retrospective Study of the Clinical Characteristics of Asymptomatic
COVID-19 Patients. mSphere: September/October 2020 Volume 5
https://msphere.asm.org/content/msph/5/5/e00922-20.full.pdf

B. R. Hoppe (1978) Blood Smears and the Use of Wright’s Stain. Vol 40: 113-116.
https://lib.dr.iastate.edu/iowastate_veterinarian/vol40/iss3/10/

L.M. Schiffer (1962) Fluorescence Microscopy with Acridine Orange: A Study of hemopoietic Cells in
Fixed Preparations. Blood. Vol. 19: 201-207

Live Blood Analysis

https://en.wikipedia.org/wiki/Live_blood_analysis

W.M. Davidson and D.R. Smith (1954) A morphological sex difference in the polymorphonuclear
neutrophil leucocytes. Brit. Med. J. 2: 6

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M.L. Barr and E.G. Bertam (1949) A Morphological Distinction between Neurones of the Male and
Female and Behaviour of the Nucleolar Satellite during accelerated Nucleoprotein synthesis. Nature
163:676-677.

G. Varricchi et al. (2018) Human mast cells and basophils how are they similar and how are they
different? Immunol. Rev., 282: 8-34 doi: 10.1111/imr.12627.

Lyme disease (Wikipedia)

https://en.wikipedia.org/wiki/Lyme_disease

C. Kurokawa et al. (2020) Interactions between Borrelia burgdorferi and ticks. Nature Reviews
Microbiology 18: 587-600.
https://www.nature.com/articles/s41579-020-0400-5

N. Aditili, M. Melizi and H. Belabbas (2016) Species determination using the red blood cells in
morphometry in domestic animals. Veterinary World 9: 960-963.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5057034/

E. Mistek-Morabito and I. K. Lednev (2020) Discrimination between human and animal blood by
attenuated total reflection Fourier transform-infrared spectroscopy. Communications Chemistry 3: 178
https://www.nature.com/articles/s42004-020-00424-8

C.R. Bagnell (2012) Darkfield Microscopy Chapt. 9 Pathology 464 – Light microscopy.
https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/Dark%20Field%20microscopy.pdf

Rouleaux Wikipedia
https://en.wikipedia.org/wiki/Rouleau

Wrights Stain purchased from Benzmicroscope.com

https://benzmicroscope.com/products/slide-stain-kits-wrights-blood-stain-2pk

J. Sprute Red Blood Cell Abnormalities in Morphology

https://hematologylearning.weebly.com/red-blood-cell-inclusions-and-abnormalities.html

Understanding your Pet’s Diagnostic Testing – Blood & Urine

https://vetclinicmission.com/wp-content/uploads/2017/12/Understanding-Your-Pets-
Diagnostic-Testing.pdf

YouTube Blood Analysis Videos

Red Blood cells viewed under the microscope in hypo and hypertonic conditions
https://www.youtube.com/watch?v=A8cI6FkcG4c

Wright and Geimsa stain for blood

https://www.youtube.com/watch?v=9xBcm-1NMq

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What blood cells look like in a Microscope
https://www.youtube.com/watch?v=uxzDrvIoh4Q

Live Blood Analysis

https://www.youtube.com/watch?v=JpWRgNJQeAo

Wright’s Blood Stain

https://benzmicroscope.com/products/slide-stain-kits-wrights-blood-stain-2pk

All About Blood Tests

https://www.healthline.com/health/blood-tests

Digital Chip Analyzes Blood

https://www.futurity.org/digital-chip-analyzes-blood-from-tiny-drop/

Looking at blood under the microscope

https://www.youtube.com/watch?v=_Hs-kUBbakU

Wrights Stain purchased from Benzmicroscope.com

https://benzmicroscope.com/products/slide-stain-kits-wrights-blood-stain-2pk

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