How to Use a Hemacytometer

Classic Techniques in Cell Biology:
How to use a hemacytometer

by Heather Buschman

French physiologist Louis-Charles Malassez (1842-1909) studied a lot of things in his life. In dentistry, the residual cells of the epithelial root sheath in the periodontal ligament are now called the epithelial rests of Malassez. A genus of fungi is also named for him, which includes species that can cause dandruff and other skin infections.

Malassez also invented the hemacytometer, the thick glass microscope slide traditionally used to count the number of cells in a given volume of liquid. He made an indentation in a regular microscope slide to form a small space that could hold a few drops of cells in suspension. With etched lines and a known area and depth, the number of cells in that particular volume could be calculated. Since the invention was first used to count blood cells, it became known as the hemacytometer (hem = blood, cyto = cells), sometimes also spelled hemocytometer or haemocytometer.

Over the past few years, more than 4,000 people viewed a Scientist Solutions discussion on how to count cells with a hemacytometer. Even for people who learned cell cultures years ago, remembering the calculation can be tricky. Here’s a quick cheat sheet:

1. Transfer a small sample of cell suspension (~100 μl) to a microfuge tube.

2. Dilute 1:2 with Trypan blue or other cell stain.

3. Carefully transfer approximately 10 μl of cells to one of the semi-reflective panels on a hemacytometer covered with a cover slip. Allow the capillary action of the cover slip to gently draw in just enough liquid to fill the chamber liquid. Do not overload.

4. Under the microscope, you should see a grid of 9 squares. Focus the microscope on one of the 4 outer squares in the grid. The square should contain 16 smaller squares.

5. Count the number of live cells in at least 2 of the outer squares. (Dead cells turn dark blue with Trypan staining.)

6. Use the following equation to calculate the number of cells in the original volume:



Some tips for best results:
• Gently pipette the cell suspension up and down to mix well and reduce aggregation before removing a small sample.

• Remove any bubbles in the hemacytometer before counting.

• Count cells touching the top and right borders of each of the 4 squares, but not the cells touching the bottom and left borders.

• If there are less than 100 cells total in all 4 squares, count more squares or start again with a more concentrated cell suspension. If there are too many cells to count accurately, start again by diluting the sample 1:2 or 1:10 before adding Trypan blue.

REPLACING MICRORNA FOR CANCER TREATMENT

Inserting a missing molecule in mice may shrink liver tumors or limit their growth By Jenny Lauren Lee Web edition : Thursday, June 11th, 2009


It’s a simple idea: Treat cancer by finding out what’s absent in a cancer cell and replacing it.

Experiments in mice suggest that inserting one small missing molecule could fight cancer without harming normal tissue, researchers at Johns Hopkins University in Baltimore and the Nationwide Children’s Hospital in Columbus, Ohio, report in a study in the June 12 Cell.

The molecule in this case is a small RNA chain known as a microRNA. MicroRNAs (abbreviated as miRNAs) are involved in a wide range of body processes relating to cellular differentiation and tissue growth. They run about 22 units long, but their size belies their influence — they help regulate hundreds of thousands of genes.

In the past decade, certain flavors of miRNA have been shown to be missing from cancer cells. Scientists have previously explored the idea that replacing the missing miRNAs might reverse the cancer, but not in a way that could lead to viable treatments, said Joshua Mendell of Johns Hopkins, one of the study’s authors.

“One of the things that distinguishes this work is that we used a clinically relevant delivery vehicle to replace a microRNA that’s missing,” Mendell says. The team used a harmless virus that acts as a sort of mail carrier to deliver the miRNA to the cells.

The researchers worked with mice made to develop tumors similar to those in human liver cancer. Most of the untreated mice developed cancers that nearly consumed their livers. But eight out of 10 mice receiving the miRNA had small liver tumors or none at all.

“It’s door-opening research” that will encourage others to move into the field, says George Calin of the University of Texas M.D. Anderson Cancer Center in Houston.

Mendell and colleagues focused on a particular strain of miRNA called miR-26a, chosen because it was absent in liver cancer cells but abundant in normal liver cells. “We thought that the tumor cells might be sensitive to the miRNA” but that the normal parts of the liver would not be harmed, Mendell says. Restoring the miR-26a stopped the cancerous cells from creating two molecules called cyclins that are used in cell replication, effectively preventing the cells from multiplying.

“Although microRNAs have been shown to play an important role in cancer pathogenesis, not too many reports have shown that altering a single microRNA can suppress cancer development in vivo,” comments Baohong Zhang of East Carolina University in Greenville, N.C. “This result has opened a novel strategy for cancer therapy.”

The study has great practical benefit, Calin says, because it shows potential for treating a specific type of cancer — hepatocellular carcinoma, or HCC, which accounts for 80 to 90 percent of all liver cancers.

Calin also says that this research suggests it may be possible to use different miRNAs as therapies for many diseases, not just cancers. MiRNAs have a wide variety of functions, and some have been linked with autoimmune disorders, diabetes and heart disease, he says.

Despite the promising results, Mendell says, there are a lot of hurdles to jump before this type of treatment could be used for people.

For one thing, the liver cancer in the mice is much more aggressive than most human liver cancers. The mouse livers were infested with multiple tumors; humans with HCC typically have only one. And since much is still unknown about how miRNAs work, this type of therapy might have unwanted consequences.

“I think we’re at the earliest stages of understanding,” Mendell says. “There’s more we don’t know than we know.”

TUBERCULOSIS BACTERIUM SUBVERTS BASIC CELL FUNCTIONS

New findings reveal that the microbe achieves virulence by disrupting immune cells' internal processes By Nathan Seppa Web edition : Wednesday, June 10th, 2009

Tuberculosis microbes invading human immune cells carry a cargo that increases TB virulence by inducing the cells to act less like sentinels and more like bystanders, tests in mice show. In a report in the June 11 Nature, a team hypothesizes that this initial infection strategy lays the groundwork for TB’s uncanny ability to lie dormant in an infected person for years.

Even though TB has been studied for hundreds of years, it still guards many secrets — including precisely how it undercuts immune cells.

“Understanding the mechanisms by which the bacteria are having their way with our host cells will be very helpful in coming up with targets that we might hit,” says Kathleen McDonough, a microbiologist at the State University of New York at Albany and the Wadsworth Center of the New York State Department of Health, also in Albany.
Mycobacterium tuberculosis gets engulfed by cells called macrophages, the shock troops of the immune system. Scientists knew that the bacteria had evolved mechanisms to proliferate in these cells, but exactly how was unclear, says coauthor William Bishai, an infectious disease physician at Johns Hopkins University in Baltimore.

Bishai and his colleagues had noted that both TB bacteria and the macrophages they had entered had excess amounts of cyclic AMP, a compound that serves as a master regulator of many cell functions. So the researchers investigated an enzyme needed to make cyclic AMP. Curiously, M. tuberculosis has 17 genes that code for versions of this enzyme.

To test the effects of these 17 genes, the researchers infected mice with TB using aerosol sprays containing various combinations of the genes. Microbes harboring the enzyme encoded by a gene called Rv0386 out-competed the other microbes for survival in the mice and caused more severe lung disease. That suggests that the gene, along with the excess cyclic AMP production it induces, helps the microbe to sabotage the macrophage’s defensive abilities, thus making the TB more virulent.

In lab tests on human macrophages, the researchers showed that M. tuberculosis gins up cyclic AMP production upon entering a macrophage and unleashes this cargo once inside. That sets off a chain reaction that steers the cells to overproduce TNF alpha, a protein that causes inflammation, which contributes to tissue damage. Mice infected with real TB made 10 times as much TNF alpha in their lungs as did mice infected with TB lacking the Rv0386 gene.

This study is the first to pin down that M. tuberculosis is actively trafficking cyclic AMP into the cells and that “this really does affect the outcome of infections,” says McDonough.This reliance on cyclic AMP and the enzyme that makes it could prove useful to drug makers in the long run, she says.

Meanwhile, excess inflammation spurred by TNF alpha may be linked to the formation of lesions called granulomas, a hallmark of TB, Bishai says. TB granulomas have a central core of dead tissue surrounded by fatty debris and an outer rim of fibrous tissue and immune cells. Granuloma formation corrals the microbe but also enables it to lie latent in a person for decades. “We hypothesize that TB has a program that leads to granuloma formation, which gives the organism sanctuary,” Bishai says.

STRESSED-OUT DNA TURNS MOUSY BROWN HAIR GRAY

Stem cells responsible for hair color lose self-renewing abilities By Laura Sanders Web edition : Thursday, June 11th, 2009

Gray hair may be a mark of distinction in some circles, but it’s also a sign of a depleted stem cell population. DNA damage causes stem cells that produce hair-color cells in mice to lose their “stemness,” leaving brown hair gray, a report in the June 12 Cell shows. The results suggest a new way stem cell populations can be depleted as cells accumulate DNA damage over time.

The new study “opens up a new paradigm for how we’re going to study stem cell aging in many systems,” comments Kevin Mills of the Jackson Laboratory in Bar Harbor, Maine. The report “fills in what’s been a hole in our understanding of stem cell biology.”

Colorful locks depend on a group of special cells in hair follicles called melanocyte stem cells. Each of these cells divides into two cells: One that replaces itself and another that differentiates into a pigment-producing daughter cell called a melanocyte, which imbues hair with its browns, reds and blacks. Earlier research has suggested that the depletion of these stem cells was to blame for grayness. But how exactly these stem cells disappeared was mysterious. With no more stem cells around to produce melanocytes, hair turns gray.

Emi Nishimura at Tokyo Medical and Dental University in Japan and her colleagues tracked the fate of these stem cells and grayness in mice exposed to DNA-damaging radiation. The exposure level was fairly high, intended to magnify the effects of DNA damage that cells gradually accumulate with age.

Mice typically begin to go gray when they are 1 year to 1½ years old, after about 65 percent of their life, Nishimura says. But following exposure to high doses of radiation, hair on mice as young as 7 to 8 weeks grew in gray, while control mice remained brown, the team found. Other DNA-damaging agents, including hydrogen peroxide, had the same graying effect.

The team next looked at the stem cells in the hair follicles during this graying process. Researchers usually think of two ways stem cells stop working, say Paul Hasty, a geneticist at the University of Texas Health Science Center in San Antonio. The stem cells either die or stop dividing, he says. “But for these melanocyte stem cells, that’s not what happens.”

Instead, the DNA damage causes them to lose their “stemness,” the new report shows. Once the cells have racked up enough DNA damage, they become melanocytes and lose the ability to replace themselves or to replenish melanocyte cell populations. Once the melanocytes die, the hair is left with no pigment-producing cells.

“What’s really unexpected is that the cells differentiate in response to DNA damage,” instead of dying or halting division, Mills says. This irreversible pathway might be going on in stem cells in other tissues like the brain or blood, he notes. Figuring out the details of how these cells lose their “stemness” may ultimately lead to new ways to stave off stem cell depletion. “If you can modulate the stemness checkpoint, you can influence the activity of these stem cells,” Mills says.

ORIGINS OF THE SWINE FLU VIRUS

Researchers use evolutionary history to trace the early days of the pandemic By Laura Sanders Web edition : Thursday, June 11th, 2009

Closely related forms of the H1N1 strain of influenza virus circulated undetected in swine for years, a study published online June 11 in Nature reports. The virus, which has spread to multiple continents, has now been classified by the World Health Organization as a pandemic.

“Based on this report, we had a virus circulating in pigs for 10 years and nobody knew anything about it because we were not doing proper surveillance,” says Daniel Perez, an influenza expert at the University of Maryland in College Park.
Researchers traced the sordid past of the H1N1 virus by comparing mutations among different strains of the virus. Genetic sequences of 15 swine influenzas from Hong Kong and two human H1N1 viruses were compared with 796 sequences representing a large spectrum of related strains from humans, birds and pigs.

Analyzing numbers of mutations allowed an international team of researchers to estimate how long ago the strains first existed. Virus strains more than 90 percent identical to the current H1N1 strain were circulating in pigs between 9.2 and 17.2 years ago, the researchers found. The current strain “evidently spread without anyone noticing it for 10 years,” says Michael Worobey, an evolutionary biologist at the University of Arizona in Tucson and one of the study’s authors. “We need to spend more energy looking at what’s in pigs.”

The molecular clock method the team used assumes that genomes mutate at relatively constant rates, a tricky assumption for sporadically mutating influenza strains.

“Any estimate like this has a certain amount of uncertainty to it,” Worobey says. Although the numbers are not exact, he says, the data clearly show that a similar version of the virus was around long before anyone was aware of it.

The report also shows that each bit of the current virus’s DNA had been circulating on its own and primarily in pigs for years before combining to form the virus responsible for the current pandemic. Some genes have been in pigs for decades. “Across the genome, this is something that came from pigs,” Worobey says.

Some of these DNA segments came from a North American swine influenza virus, which itself is made of bits of avian, human and swine influenzas (called a triple-reassortant strain). Other segments came from Eurasian swine with avian virus components. The combination of the triple-reassortant strain from North America and the avianlike strain from Eurasia probably happened as live pigs were transported between North America and Eurasia, the authors say.

“We can do all the surveillance we want in humans, but if we really want to prevent pandemic influenza…, a fundamental change in efforts on the animal health side has to be made,” Perez says.

On the same day the new report appeared, the World Health Organization classified the H1N1 outbreak as a pandemic, defined as showing sustained person-to-person transmission in many parts of the world.

WHO Director-General Margaret Chan said that the organization is raising the alert level after determining that flu cases are now showing up in people who didn’t bring it from another region and weren’t in contact with such travelers. “Further spread is considered inevitable,” Chan said in a news conference.
Thomas Frieden, director of the Centers for Disease Control and Prevention, said in a June 11 press briefing that the new classification does not imply that the H1N1 virus has become more virulent.

“This does not mean that there is any difference in the level of severity of the flu,” he said. Rather, the pandemic label “is important because it does send the strong message that the virus is here, it’s in all likelihood here to stay, and it’s important that we continue our aggressive efforts to prepare and respond.”

So far no decision has been made to mobilize pharmaceutical companies to start mass-producing vaccines aimed specifically at the novel H1N1 virus. But preliminary steps to make that a seamless move have already been taken.

PLUMP YOUNGSTERS SHOW HEART-Y RISKS

By Janet Raloff Web edition : Friday, June 12th, 2009

Blood markers observed in obese children — some as young as 7 — indicate their bodies host chronic inflammation, a driver of heart disease, and elevations in chemicals that promote blood clots.

The findings, reported today at the Endocrine Society annual meeting, in Washington, D.C., indicate that school-age plumpness can prove more than a social stigma. It may signal that youngsters are on their way to developing cardiovascular disease — and years earlier than even a generation ago.

Nelly Mauras, chief of endocrinology at Nemours Children’s Clinic in Jacksonville, Fla., and her colleagues sampled the blood of 202 children aged 7 to 18. Half were lean and came from healthy, lean families. The rest were obese, but not due to disease. Moreover, for inclusion in the new study, heavy kids could not have metabolic syndrome, a condition whose constellation of symptoms — from high blood pressure and high cholesterol to elevated blood sugar — marks individuals at risk of heart disease, stroke and diabetes.

With childhood obesity reaching epidemic proportions, Mauras’ group wanted to see whether extreme pudginess in youngsters posed the same potential heart risks as it does in adults. And her disturbing data now indicate that it indeed appears to.

C-reactive protein is a blood marker of systemic inflammation. Concentrations of this CRP were, on average, eight times higher in fat adolescents (those who had at least entered puberty) than lean kids their age. And for kids who had not yet entered puberty, CRP values were 12 times higher among the very overweight kids, compared to lean children their age. Not surprisingly, the heavy kids also had elevated levels of interleukin-6, a signaling molecule that stimulates the liver to make CRP.

Adiponectin is a hormone secreted by fat cells that possesses anti-inflammatory properties. Its blood level was lower in the heavy kids than their lean counterparts, Mauras reports, while values of PAI-1, a compound that inhibits the breakdown of blood clots, were notably higher in overweight youngsters. Heavy kids also had significantly higher blood concentrations of fibrinogen, a material that fosters blood clotting.

At a briefing for reporters, Mauras concluded that “the unhealthy consequences of excess body fat start very early in childhood.”

“That’s kind of scary, isn’t it?” asked Daniel Bessesen, chief of endocrinology at the University of Colorado Denver School of Medicine, after hearing the new findings. “We know that cardiovascular disease develops not over a year or two but over or 20 and 30 years. And if kids have that kind of biochemical profile when they’re 7 to 9 years of age, you wonder how our country’s going to deal with that 30 or 40 years from now. “

Indeed, will we be in the throes of a middle-age heart-disease and stroke epidemic? And if health care costs are almost beyond our means to manage now, how much worse will they become?

Mauras didn’t go into what’s behind childhood obesity, but plenty of nutritionists point to one growing problem: Too little exercise. Elementary and secondary schools around the country have been eliminating physical education programs. Aggravating the problem, those computers and video games that well-meaning parents have been bringing into our homes have been seducing kids into becoming couch potatoes.

Kids may not know the long term implications of a sedentary lifestyle. But we parents do — and need to get the nation’s youngsters up and moving again.

Link between mammal, reptile, and bird

Check out this long beaked echidna! A very cool article on this wonderful animal just came out in the New York TImes. I had never heard of such a creature. It is a cousin to the platypus and has a fantastically large brain. (No wonder it avoids all human contact. It probably would have been hunted out of existence.) There is also a link to the very first field study on this animal in the Journal of Mammalogy.
Here are a couple of cool picks from the New York Times.

Chromosomes and Cytogenetics Topic Room

Cytogenetics is the study of chromosomes and their role in heredity. Thus, this topic room is all about chromosomes: chromosome structure and composition, the methods that scientists use to analyze chromosomes, chromosome abnormalities associated with disease, the roles that chromosomes play in sex determination, and changes in chromosomes during evolution.

The field of cytogenetics emerged in the early twentieth century, when scientists realized that chromosomes are the physical carriers of genes. As is always the case in science, researchers built on the observations of their fellow investigators to synthesize the chromosome theory of heredity. This groundbreaking theory had its foundations in the detailed observations that cytologists had made about chromosome movements during mitosis and meiosis, which suggested that chromosome behavior could explain Mendel's principles of inheritance.

In the early years of cytogenetics, scientists had a difficult time distinguishing individual chromosomes, but over the years, they continued to refine the conditions for preserving and staining chromosomes to the reproducible standard that is now expected in clinical cytogenetics. (Looking back, it seems incredible that the human chromosome number was not established until 1955.) In today's procedures, metaphase chromosomes are treated with stains that generate distinctive banding patterns, and chromosome pairs are then arranged into a standardized format known as a karyotype. Among the members of a species, karyotypes are remarkably uniform, which has made it possible for cytogeneticists to detect various deviations in chromosome number and structure that are associated with disease states and developmental defects.

A normal human karyotype contains 22 pairs of autosomes and one pair of sex chromosomes. Aneuploidies, or changes in chromosome number, are easily detected on karyotypes. In humans, most aneuploidies are lethal because of the ensuing imbalance in gene expression. A notable exception is trisomy 21, or Down syndrome, which is frequently detected during prenatal screening of older mothers. Sex chromosome aneuploidies are also tolerated in humans, most likely because X inactivation maintains near-normal expression levels for X-linked genes. In addition to changes in chromosome number, karyotypes can also reveal more subtle changes in chromosome structure. In effect, the normal banding pattern of a chromosome provides a "bar code" that can be translated into a map of the chromosome. Cytogeneticists can then use coordinates on these rough chromosome maps, or idiograms, to identify the positions of structural abnormalities, including deletions, duplications, and translocations, to within a few megabases of DNA.

Over the past few decades, versatile methods based on fluorescence in situ hybridization (FISH) have transformed cytogenetics into a molecular science and provided cytogeneticists with powerful new tools. In FISH procedures, labeled DNA or RNA probes are hybridized with their complementary target DNA sequences on chromosomes. FISH experiments often generate colorful results, because multiple probes, each of which is labeled with a spectrally distinct fluorescent dye, can be used in the same experiment. The target DNA sequences may consist of either a single gene or a collection of genes spread out along the length of a chromosome. FISH procedures are now routinely employed in clinical cytogenetics. Spectral karyotyping provides an overview of any gross rearrangements and changes in chromosome number that have occurred in a patient's cells. Using gene-specific probes, cytogeneticists can also positively identify the genes affected by chromosomal mutations. More recently, researchers have additionally begun to employ comparative genomic hybridization to analyze small quantitative differences between individuals' DNA, including copy number variations (CNVs).

Outside the clinic, FISH is one of many techniques biologists use to investigate the structure of chromosomes and their organization within the nucleus. Although chromosomes may appear to be static structures when viewed under a microscope, cytogeneticists know that chromosomes are actually dynamic assemblies made up of a DNA-protein complex called chromatin. Chromatin undergoes dramatic changes in packing during the cell cycle, and its structure also varies locally along the length of each chromosome. Transcriptionally active chromatin, or euchromatin, has a different composition than silent chromatin, or heterochromatin. (The inactive X chromosome in female mammals is a special case in which heterochromatin extends along the entire length of a chromosome.) Some chromatin specializations are essential for normal chromosome behavior. For example, centromeres contain a unique chromatin that is required for chromosome attachment to the mitotic spindle. Likewise, chromosome integrity depends on the assembly of a specialized chromatin found exclusively at the telomeres. Other less defined aspects of chromosome structure may also be important in positioning individual chromosomes with the nucleus. For instance, mounting evidence seems to indicate that chromosomes occupy discrete territories in the interphase nucleus; this marks a significant departure from the previously accepted idea that chromosomes are randomly organized during interphase.

In this era of comparative genomics, cytogenetics is also offering insights into evolution. Using cross-species FISH, scientists have identified groups of genes, called synteny groups, that maintain the same linkage relationships with each other across species boundaries. Synteny data reveal numerous chromosomal rearrangements that have occurred during the course of evolution. Taken together with DNA sequence information, synteny data are proving useful for detecting genome duplications and for constructing phylogenetic trees.

The collection of articles in this topic room is intended to provide students with an introduction to chromosome biology and an appreciation of the experimental evidence that has led to the current state of understanding. Cytogenetics is a broad and growing field of research, and many topics have not been discussed in detail. The editors hope that this collection will grow over time as new discoveries are made and gaps in the current collection are filled. To this end, teachers and researchers are encouraged to contribute new articles to the collection after consultation with the editors.