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68% of measurements are found within one standard deviation σ from the mean μ; 95% are found within
1.96 times the standard deviation from the mean.
Example
A population of liver cells has p.H. with a mean of 5.61 and a standard deviation of 0.33. What are the
upper and lower limits between which 95% of the cells are found? If a cell has a p.H. of 6.10 is it unusual?
Solution
1) 95% of cells will be within 5.61 ± (1.96 x 0.33) = 5.61 ± 0.65 = between 4.96
and 6.26
2) A p.H. of 6.10 is well within these limits so it is not unusual.
The Estimate of the Standard Deviation: We divide by N -1 and not N because we set the mean to be in
the middle of the sample. It might not, in fact be there, and so using N tends to underestimate the
standard deviation. N -1 is called the degrees of freedom
The mean of a sample is likely to vary (but by less than individuals) because you may have picked by
chance samples with more high values of the measurement than average or more low values. The
variability of the estimate of the mean is easily calculated and is called the standard error of the
mean, SE, which is given by the expression: SE = s /√N
Graphing data with error bars. (a) The mean yield of two species of grass with error bars showing their
standard deviation; this emphasises the high degree of variability in each grass, and the fact that the
distributions overlap a good deal. (b) Standard error bars emphasise whether or not the two means are
different; here the error bars do not overlap, suggesting that the means might be significantly different.
However, from the population we have sampled we are not sure of the standard error, SE. All we have is
the estimate, SE, which might be larger or smaller than the real value.
The t Distribution
For this reason the mean has a broader distribution relative to SE called the t distribution. This is
especially wide when the sample size (and hence the number of degrees of freedom, N -1) is low.
However, it approaches the normal distribution when the sample size is high and hence when the
estimated standard error SE approaches that of the population SE.
Normal distribution and t distribution. The distribution of sample means x relative to the estimate of the
standard error SE calculated from samples with 1, 10 and infinite degrees of freedom. With infinite
degrees of freedom the distribution equals the normal distribution. However, it becomes more spread out
as the sample size decreases (fewer degrees of freedom) because the estimate of standard error
becomes less reliable.
T TESTS
You may want to ask several different sorts of questions about the measurements you have taken.
Is the mean different from an expected value?
Are the means of two measurements made on the same sample different from each other?
Are the means of two separate samples different from each other?
Is the mean of an experimentally treated group different from that of the control?
To answer these questions you need to carry out statistical tests for differences - t tests.
The question is how different must the sample mean be from a particular value, E, to make it unlikely
that the population mean is E?
Figure 1
There's a high probability of getting a mean, x, at least 1 x SE away from the real mean, μ.
There's a very low probability of getting a mean, x, at least 3 x SE away from the real mean, μ.
Compare the absolute value of t, |t| , with the critical value for 5% with N - 1 degrees of freedom. (N =
sample size).
If |t| < tcrit , then there is a greater than 5% probability of this happening by chance. Therefore you have
no evidence to reject the null hypothesis. However if |t| ≥ tcrit there is a less than 5% probability of this
happening by chance. Meaning that you do have evidence to reject the null hypothesis and you can say
the mean is significantly different from the expected mean, E.
Example
In our data for the sodium ion concentration of a sample of 9 cells we found that the mean, x = 0.86 M,
standard deviation, s = 0.11 M, and standard error, SE = 0.037 M.
A textbook says that the concentration in these cells is 0.93 M. Do your cells have a significantly different
mean sodium ion concentration than expected?
Solution
The null hypothesis is that the sodium ion concentration is really the expected value, 0.93.
It is easy to calculate t
Looking in the table for critical values of t, tcrit for 5% at (9 - 1 = 8) degrees of freedom = 2.306
4) 1.89 < 2.306. Therefore there is a greater than 5% probability of this happening by chance. We have
no evidence to reject the null hypothesis. Our cells don't have a significantly different mean sodium
concentration than expected.
Mark 78 82 4
Example
The Effect of Coffee on Heart Rate: Brian 60 59 -1
1) The Calculation of Mean Difference
Mean Difference, d = 4.2 Paul 45 50 5
Standard Deviation of difference, sd =
3.0 Angus 55 59 4
Standard Error of difference SEd =
1.01 Richard 96 97 1
Peter 51 54 3
Patrick 64 70 6
It looks like people have on average a higher heart rate after drinking coffee, but is this a significant
difference?
Now for the t test itself
2) The t test
The null hypothesis is that the mean difference before and after taking coffee is zero
Calculate t:
3) There are 9 people, so there are 8 degrees of freedom. The critical value of t for 5% at 8 degrees of
freedom t8 = 2.306.
4) 4.16 > 2.306. Therefore there is a less than 5% probability of this happening by chance.
We can say, therefore, that drinking coffee significantly alters heart rate (it increases it, actually).
Testing for Differences between two Groups
In Biology it is much more common to have to see if two groups have measurements with different mean
values.
Examples
Control organisms and experimentally treated ones, males and females of the same species, ntracellular
and extracellular fluid, cells from two different parts of the body. This is difficult because both samples
vary.
To tell what the cut-off is we perform a 2 sample t test. The Two Sample t Test
Purpose: to test whether the mean values of a measurement made on two separate samples are
different from each other.
Rationale: you calculate how different the means of the two samples are.
You compare that with a measure of how variable the difference between the means of the two samples
is.
a) If the error bars do overlap, the means are not significantly different.
b) If the error bars do not overlap, the means may be significantly different.
Example:
Comparing the Longevity of Control and Irradiated Worms
Samples of 16 worms were taken from each group. The results are summarized below.
Control Irradiated
Solution
1) The null hypothesis is that there is no difference between the longevity of the two groups.
2)
3) There are 16 worms in each group, so there are (16 + 16 - 2 = 30) degrees of freedom. The critical
value of t for 5% at 30 degrees of freedom t30 = 2.042.
4) 0.63 < 2.042. Therefore there is a greater than 5% probability of this happening by chance.
We have no evidence to reject the null hypothesis. Irradiated and control worms do not have
significantly different longevity.
Types of association
If you take 2 measurements on a single sample you can investigate two different things.
1) Whether the mean of the two measurements is different
eg Is mean heart rate different before and after drinking coffee?
We have seen you can use the paired t test to do this.
2)Whether the two measurements are associated
eg. Do people with high blood pressure also have high heart rates?
In a small sample (a), it's quite likely you can get points that seem to show an association.
In a large sample (b), it's very unlikely you would choose points that all fitted along a straight line.
To determine whether an association is likely to be real, therefore, you need to carry out a statistical
test.
Stage one is to work out the correlation coefficient.
Association (r = -1)
Perfect Positive
Association (r = 1)
No Association (r = 0)
The further r is from 0, and the larger the sample size, the less likely is the association due to chance.
Example
An investigation seeks to find if brain size of monkeys is related to the size of the gut. The following
results are obtained.
Brain Mass Gut Mass
Species
(% body mass) (% body mass)
1 6 25
2 8 20
3 9 18
4 10 13
5 12 14
Is there any association between the two measurements?
Solution
The first stage is to calculate x and y.
Mean brain mass = 9%
Mean gut mass = 18%
Calculating r
Σ(x - x)(y - y) = (-3 x 7) + (-1 x 2) + (0 x 0) + (1 x -5) + (3 x -4)
= -21 - 2 - 5 - 12
= -40
Σ(x - x)2 = (-3)2 + (-1)2 + (0)2 + (1)2 + (3)2
=9+1+1+9
= 20
Σ(y - y)2 = (7)2 + (2)2 + (0)2 + (-5)2 + (-4)2
= 49 + 4 + 25 + 16
= 94
Therefore
= -40/43.36 = -0.922
Examples
a) If you are measuring cell mass at different times.
b) If you are controlling one variable yourself in an experiment (eg if you are measuring metabolic rate of
cells at certain temperatures).
2) The fact that there is a correlation does not necessarily imply a causal relationship between the two
sets of measurements.
eg In our example brain mass does not control gut mass or vice-versa. In fact the negative correlation is
related to the diet of different monkeys. Leaf-eating monkeys have larger guts to digest the leaves, but
do not need as big brains as fruit-eating ones because leaves are more plentiful and easier to find than
fruit.
3) The size of the correlation coefficient does not reflect the slope of the relationship, just its closeness.
4) Correlation is only useful for linear relationships, so always look at graphs of your results, first.
CHI Square Tests
Categorical Data: There are many characteristics of organisms that cannot be measured to give numbers
(eg 3.41). Instead the characteristics can only be put into categories.
Eg. Gender (Male/Female), Infection (Present/ Absent), Colour (Red, Yellow, etc), Species (Thrush,
Dunnock, etc)
These can only be quantified by counting the frequency with which each character state turns up. We
might then ask two sorts of questions.
Are the frequencies of particular characteristics different from what we might expect?
Are certain characteristics associated with one another?
Rationale
To work this out you work out the X2 statistic. This is found by the formula
O = Observed Value
E = Expected Value
The further your values are from the expected ones the bigger is X2 and the less likely the results are to
have happened just by chance.
3) Compare X2 with the critical value for 5% significance with N - 1 degrees of freedom, (N = the number
of groups)
4) If X2 < X2crit therefore there is a greater than 5% probability of this happening by chance. You have no
evidence to reject the null hypothesis. If X 2 ≥ X2crit therefore there is a less than 5% probability of this
happening by chance. You do have evidence to reject the null hypothesis. You can say that the
frequencies are significantly different from the expected values.
Example
The incidence of malaria in a region where a new insecticide had been applied was 320 people out of a
population of 15,000. This compares with an incidence in the country as a whole of 3%. Has the
insecticide made a significant difference on malarial infection?
Solution
1) The null hypothesis is that the region has the same incidence of malaria as the country as a whole.
2) The first stage in working out X2 is to calculate the expected numbers of cases. Expected no. cases =
15,000 x 0.03 = 450
(Expected no. of fit people = 15,000 - 450 = 14,550
= (-130)2/450 + 1302/14,450
= 37.56 + 1.17 = 38.73
3) There are two groups (fit and ill) therefore 2 -1 degrees of freedom. X21 = 3.84.
4) 38.73 > 3.84 therefore there is a significant difference in distribution. In fact there are fewer cases
than expected
And put the results (in brackets) into the Contingency Table:
ANOVA
Often you might want to carry out rather more complex surveys or experiments. You might want to
compare 2 experimentally treated groups of rats with a control group. You might want to compare
people who have been put on 4 different drug regimes.
You might want to compare 5 strains of bacteria. You might think you could carry out pairwise t tests, eg
1 vs 2, 1 vs 3, 2 vs 3
But there are two problems, you would need to do lots of tests
Number of Groups 3 4 5 6 7 8 9 10
Number of Tests 3 6 10 16 23 31 40 50
Each time you perform a test there is a 5% probability of getting a significant answer by chance alone.
Therefore the more tests you do the more likely you are to get a wrong answer.
For this reason you must use a different test: Analysis of Variance or ANOVA
2) Calculating the Variance or Mean Squares. This is done by dividing by the correct numbers of
degrees of freedom.
a) Within Sample "Mean Square", MSw
Where n = total number of points. N = number of groups
b) Between Sample "Mean Square", MSB
Regression
The bjectives of this Learning module are to test whether an apparent linear relationship between two
variables is real, or whether it could have happened by chance because of variability. To do this one must
use “Regression Analysis”. In an earlier unit we saw how correlation allows us to test for relationships
between two measurements taken on the same items, both of which might be dependent on the other,
eg Heart rate and blood pressure. However, there are many cases when one measurement is clearly
independent of the other one, Eg Age and Mass of people: age is clearly independent of mass. Time and
pH of cells: you look at the pH of cells at particular times. Reaction rate and temperature: you control the
temperature and this affects the rate of reaction.
You can't investigate these cases using correlation. Instead you must plot the data correctly and use
regression analysis.
Compare t with the critical value for N - 2 degrees of freedom, where N = number of points. Here tcrit =
2.069
0.247 < 2.069, so the difference is not significant. Initial weight is not significantly different from 90 g